CA2378477A1

CA2378477A1 - Induction of immune tolerance

Info

Publication number: CA2378477A1
Application number: CA002378477A
Authority: CA
Inventors: David John Roberts; Britta Christina Urban
Original assignee: Individual
Current assignee: Oxford University Innovation Ltd
Priority date: 1999-06-30
Filing date: 2000-06-30
Publication date: 2001-01-11
Also published as: WO2001002005A2; GB9915311D0; EP1196543A2; AU5695200A; WO2001002005A3

Abstract

Methods and compositions for the induction of immune tolerance in mammalian antigen presenting cells such as dendritic cells, macrophages, monocytes and B-lymphocytes are described. Such methods and compositions involve the use of agonists of the cell surface receptors CD36, CD51, thrombospondin receptors and/or the .beta.-integrins which when exposed to an antigen-presenting cell such as a dendritic cell are able to inhibit maturation therein. Thus, the cells' ability to promote an immune response is inhibited. Tolerance to a specific antigen can be induced in antigen-presenting cells by exposure to one or more of the aforesaid agonists and the antigen. Thus, cell preparations can be prepared for administration to humans where tolerance to a specific antigen or antigens needs to be induced, for example in the case of allograft or xenograft transplants or in autoimmune disease.

Description

INDUCTION OF IMMUNE TOLERANCE
The invention relates to the field of immune suppression and, in particular, to the identification of molecules which act as agonists of the cell surface receptors CD36, CD51 and thrombospondin receptors expressed on mammalian dendritic cells and other antigen-presenting cells, to ex vivo and in vivo uses of such molecules for inducing peripheral immune tolerance in mammals, to identification of molecules which inhibit the state of immune tolerance induced in a human by the binding of red blood cells infected with the malarial parasite to dendritic cells and to in vivo uses of such molecules in treating malaria.
Dysfunction of the immune system has been shown to play a role in the initial development and further progression of many human diseases. Impaired immune function can result in inability to fight infection or to destroy malignant cells as they develop within the body. Other diseases are caused because the immune system mounts an inappropriate response to a particular antigen. This inappropriate response might be to an external antigen resulting in atopic disease such as hay fever, asthma, eczema, coeliac disease and . the like or to the body's own antigens resulting in auto-immune disease. For example both the non-organ specific auto-immune diseases, such as systemic lupus erythromatosis and rheumatoid arthritis and the organ specific auto-immune diseases such as auto-immune haemolytic anaemia and idiopathic thrombocytopenic purpura are associated with an inappropriate T-cell response to selfrantigens.
Other auto-immune diseases where the antigen has been defined include auto-immune connective tissue syndromes, insulin dependent diabetes mellitus and auto-immune thyroid disease. Diseases where the antigen is less well defined include auto-immune skin diseases such as eczema, psoriasis, alopecia areata and vitiligo, auto-immune diseases of the gastro-intestinal system such as inflammatory bowel disease and auto-immune hepatitis, auto-immune diseases of the nervous system such as multiple sclerosis and myasthenis gravis and auto-immune diseases of the kidney such as glomerulonephritis.
In view of the diseases associated with inappropriate immune response, particularly T-cell response, it is highly desirable to develop pharmaceuticals which are able to damp down certain of the body's immune defence mechanisms in order to alleviate the distressing symptoms associated with these diseases.
As well as treatment of diseases specifically associated with a mal-function of the immune system, down-modulation of immune mechanisms is desirable in circumstances where a recipient is exposed to allo-antigens or xeno-antigens for therapeutic purposes such as recipients of allogeneic or xenogeneic transplants. An allogeneic response in the case of allogeneic bone marrow transplantation or donor lymphocyte infusion might be avoided if one could induce a state of peripheral immune tolerance against donor cells in the recipient. Other examples of situations where down-modulation of immune mechanisms might be desirable include haemolytic disease of the new born, neo-natal allo immune thrombocytopenia or the therapeutic administration of antigenic substances such as blood products e.g. factor VIII, or any other therapeutic or prophylactic agent likely to induce an unwanted cellular immune response.
A cellular immune response is mediated by T-lymphocytes which are activated by antigen presenting cells, the most important of which are dendritic cells, which present antigen and activate memory T-cells and naive T-cells. Dendritic cells become potent antigen-presenting cells when exposed to an immune stimulus and thereafter are described as armature". Maturation confers enhanced ability to stimulate T-cells and a reduction in pinocytosis and phagocytosis compared with immature cells.
Furthermore, maturation is accompanied by enhanced cell surface expression of HLA Class I and class II
molecules as well as adhesion molecules, including CD54 and co-stimulatory molecules such as CD80, CD86 and the cell-surface marker CD83. Maturation of dendritic cells is also accompanied by the secretion of cytokines such as TNFa and IL12p70. The secreted cytokines have an autokrine effect on dendritic cell maturation itself and parakrine effects on interacting T-cells.
Immature dendritic cells present the cell surface antigens CD36 and CD51 (a~) (part of the vibronectin receptor a~~i3) . CD36 and integrin heterodimers a"(33 or a~(35 can be cross-linked by the soluble bridging molecule thrombospondin (TSP). Through studies of malarial infection the present inventors have discovered that dendritic cell maturation on exposure to an immune stimulus, for example, lipopolysaccharide (LPS), can be inhibited by molecules which bind to CD36 or to CD51 or both via the bridging molecule TSP
and which act as agonists thereto.
This discovery is based on the inventors' initial observations that red blood cells infected with the material parasite Plasmodium falciparum adhere to dendritic cells via CD36 and/or TSP/CD51 (see Figure 1) and are able to.inhibit the maturation thereof on exposure to LPS.
Plasmodium falciparum is one of the most successful human pathogens for which virulence factors remain poorly defined, although adhesion of infected erythrocytes to venular endothelium has been associated with some of the symptoms of severe disease. Immune responses are unable to prevent symptomatic infections throughout life and immunity to severe disease develops only slowly during childhood.
Understanding the obstacles to the development of protective immunity is crucial for rational approaches to prevent the disease.
Specific immunity to malaria has been attributed to cytotoxic lymphocytes active against the liver stage of infection or to antibodies reacting against blood stage antigens. Antigenic diversity, clonal antigenic variation and T-cell antagonism may contribute to evasion of the protective and parasiticidal host responses.
Furthermore, it is known that Plasmodium falciparum-infected erythrocytes adhere to endothelial cells and it has been widely assumed that this adhesion has evolved to mediate sequestration of parasites to endothelial cells in the peripheral tissues and so reduce their destruction by splenic macrophages.
The present inventors have now identified a further mechanism by which the malarial parasite prevents the infected host from mounting an effective immune response and preventing recurrence of the disease.
Specifically, the inventors have observed that human erythrocytes which are infected with Plasmodium falciparum are capable of adhering to human dendritic cells and that immature dendritic cells exposed to infected erythrocytes are no longer able to mature into full antigen-presenting cells or to stimulate T-cell proliferation, when subsequently exposed to an immune stimulus. However, this state of immune tolerance is not observed when the dendritic cells are exposed to uninfected erythrocytes, uninfected erythrocyte lysate, infected erythrocyte lysate, parasite-conditioned medium or a crude pigment preparation derived from infected erythrocytes.
Further, the effect is not observed when dendritic cells are exposed to erythrocytes infected with a Plasmodium falciparum strain T9/96 which is known not to be able to adhere to endothelial cells (Gardner et al (1996) Proc. Natl. Acad. Sci. USA 93 pp 3503-3508).
This particular strain is not able to induce expression on the surface of infected erythrocytes of the parasite-derived protein pf-EMP-1 which is known to undergo clonal antigenic variation and is thought to be the mediator of adherence to endothelial cells.
It has been reported that most parasite lines and clones adhere to the known cell-surface receptors CD36 and via TSP to CD51/61 (a~(33) . It is also known that pf-EMP-1 can bind to CD36. (see WO 96/33736).
The present inventors have now shown that CD36 and CD51 influence the process of dendritic cell maturation and that agonists thereof, including the malarial parasite derived protein pf-EMP-1, antibodies specific for CD36 and CD51, negatively charged phospholipids and apoptotic cells, are able to inhibit dendritic cell maturation in response to an immune stimulus. These agonists reduce the ability of the dendritic cells to stimulate T-cell proliferation in response to an antigen to a level which is lower than cells which have not been exposed to an immune stimulus at all. Thus, agonists of CD36 and CD51 can induce a state of immune tolerance.
It follows that agonists of CD36 and CD51 would be useful for the treatment of the types of autoimmune disease described above where an over-reaction of the host immune system is responsible for the symptoms.
Further the inventors have found that dendritic cells may be treated by CD36 and/or CD51 agonists in vitro together with an antigen specific to the immune-response manifested in the auto-immune disease in question. Thus, tolerance may be induced to a specific antigen so that, when the dendritic cells are reintroduced into the host, further auto-immune reaction is avoided or substantially reduced. In addition CD36 and CD51 agonists are useful for inducing a state of immune tolerance in both host and donor dendritic cells where bone marrow transplantation or lymphocyte infusion is contemplated. The feasibility of such treatment is demonstrated herein in vivo in mice. The ability to inhibit maturation of dendritic cells can be demonstrated in vitro so that molecules which act as CD36 or CD51 agonists can be easily identified in a high throughput screening assay.
_As used herein the term "agonist" means a composition, molecule, cell or a component thereof which induces the same response when interacting with a receptor as the naturally-occurring ligand for that receptor.
In accordance with a first aspect the invention provides a method of identifying a molecule which is an agonist of cell surface receptor CD36 and/or CD51 as expressed by mammalian dendritic cells which method comprises:
a) exposing immature mammalian dendritic cells to the molecule to be tested, b) exposing said immature dendritic cells to an immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, wherein impaired maturation in response to the immune stimulus is an indication that said molecule under test is a CD36 and/or CD51 agonist.
Preferably, the method is performed using human dendritic cells. As used herein the term dendritic cells means cells that present antigen to and activate lymphocytes and which are distinguished by their ability to activate, not only memory T-cells but also naive T-cells. Dendritic cells for use in the method of the invention may be derived by cultivation of adherent peripheral blood mononuclear cells with the addition of Granulocyte-Macrophage Stimulating Factor and Interleukin-4 for about 6 to 10 days. Such dendritic cells can be characterised by their level of expression of the cell-surface markers HLA Class L and II (high), CD11 c (high), CD3 and CD19 (negative), CD14 (low) and CD86 (high). These markers distinguish them from B-cells which are positive for CD19, T-cells which are positive for CD3 and macrophages which are CD14 high and CD86 low. (See Banchereau et al, (1998) Nature 392, 245-252). Antibodies to HLA Class I, HLA
class II, CD14, CD3, CD19 and CD86 useful for identifying immature dendritic cells are commercially available as indicated in Table 1 below.
Dendritic cells which may be used in the method of the invention can also be derived directly from circulating peripheral blood mononuclear cells or by culture of CD34+ stem cells as described by Caux et al (1996) J. Exp. Med. 184:695-706 and Arrishi et al (1999) Blood 93:2244-2256.
There are various ways in which maturation of dendritic cells in response to an immune stimulus, may be measured. On maturation the dendritic cells become potent antigen presenting cells. As aforesaid maturation is accompanied by enhanced cell surface expression of HLA Class I and II molecules such as HLA
DR, adhesion molecules such as CD54 and co-stimulatory molecules such as CD40, CD80, CD86 and CD83 which is a specific marker for mature dendritic cells. Thus, examination of the cell's antigen presenting ability, for example variety of antigens and/or level of - g -expression, is one way of determining whether maturation has occurred or whether it has been inhibited by the test molecule. Preferably, following immune stimulation, the level of expression of the HLA
Class I and II molecules and/or adhesion molecules and/or co-stimulatory molecules is measured. In one embodiment maturation of dendritic cells is detected by measurement of the level of expression of two or more of the cell-surface antigens HLA DR, CD54, CD40, CD83 and CD86 whose level of expression is particularly enhanced. Preferably, the level of expression of all of the above in response to an immune stimulus is measured. Optionally the expression level of CD80 may also be measured.
Methods by which the expression of a cell-surface antigen may be quantified are well-known to those skilled in the art. The commonly used method is to apply an antibody specific for the antigen in question to the antigen-presenting cells which has been labelled to give a quantifiable detectable signal.
Suitable labels are well-known to those skilled in the art and include radioactive labels, enzyme labels, fluorescent labels, metallic particles and the like.
Antibodies suitable for carrying out the screening method of the present invention, as well as a commercial source, are shown in Table 1 below:

- g _ Anticten Antibody Source HLA DR BF-1 Serotec HAL Class 1 W32/6 ATCC HB-95 CD14 Tiik4 DAKO

CD54 6.5B5 DAKO , CD40 LOB7/6 Serotec CD80 BBl or DAL 1 Serotec CD83 HBlSa Serotec CD86 BU63 Serotec CD36 clone 89 Serotec clone SMc~ Immunocontakttec Serotec: 22 Bankside, Station Approach, Kidlington, Oxford, UK DAKO Ltd: 16 Manor Courtyard, Hughenden Avenue, High Wycombe, Bucks HP13 5RE
Immunokontakt: Centro Nord-Sud, CH-6934 Bioggio, Switzerland, Peprotec: 23 St. James Square, London SW9Y 4JH, UK, ATCC: 10801 University Boulevard, Manassas, VA 20110-2209; USA, Sigma: Sigma Alderich Company Ltd: Fancy Road, Poole, Dorset, BH12 4QH, UK, Schering-Plough: Schering-plough House, Shire Park, Welwyn Garden City, Herts,.AL7 1TW.
As an alternative to measuring the level of cell surface antigen to determine whether or not dendritic cell maturation has occurred, it is possible to measure the cell's ability to induce T-cell proliferation. This is inhibited by agonists of CD36 or CD51. Dendritic cells which have been exposed to the molecule to be tested and to an immune stimulus may be exposed to T-cells, for example allogeneic lymphocytes in a mixed lymphocyte reaction (MLR)with the T-cell receptor. The T-cells respond by growing and dividing, something which can easily be measured using methods well-known to one skilled in the art.
For example, growth and division can be assessed visually using a light microscope to observe clumps of dividing cells. Alternatively, cell proliferation can be quantified using a suitably labelled metabolite, for example tritiated thymidine, which is incorporated into the cell's DNA.
A yet further alternative for determining the degree of dendritic cell maturation is to measure the level of secretion of cytokines such as TNFa, IL2p70 or IL10.
For example IL12p70 is secreted by mature cells but not by immature cells. The level of TNFa secretion is reduced in immature as opposed to mature cells. Kits are commercially available for detection and quantitation of all of the above cytokines. (see Examples). Preferably, the levels of TNFa, IL12p70 and IL10 secretion are measured.
In the screening method of the invention a variety of immune stimuli may be used. Suitable examples are lipopolysaccharide (available from Sigma), TNFa (available from Peprotec) and monocyte conditioned medium (MCM)the preparation of which is described by Romani et al (1996) J. Immunol.
Methods, Sep 27; 196(2):137-51. Another suitable immune stimulant is CD40L which is expressed from plasmids having the ATCC Accession No's 79812,79813,79814 or 79815. The plasmids may be expressed in mouse fibroblasts STO (ATCC-CRL-1503).
It will be understood that TNFa would not be used as both immune stimulant and indicator of cell maturation in the same assay.
In a particular embodiment of the method of the invention immature dendritic cells (about 106) are exposed in duplicate to various concentrations of the test molecule for about 3 to about 12 hours in a multiwell plate. The test compound is prepared in a suitable diluent which is not toxic to the dendritic cells such as tissue culture medium, PBS, water or a suitable non-toxic organic solvent, if appropriate.
The duplicate wells are subsequently exposed to LPS
(about 500 ng/ml) or left untreated for about 48 hours. For each concentration of the compound and time of exposure, the surface expression of the molecules identified above is compared with the surface expression on immature dendritic cells exposed to the test compound as well as untreated immature dendritic cells. The increase in cell surface expression is evaluated using indirect immunofluorescence and FACScan analysis. A compound is a candidate for further evaluation if the surface expression on dendritic cells of at least two cell-surface antigens is not increased by addition of the immune stimulant, LPS.
Preferably, molecules identified as potential CD36 or CD51 agonists by the method of the invention will be subject to further evaluation. For example, if surface expression of lineage-specific molecules has been used to determine the degree of maturation it would be usual to check whether the compound can also prevent immune-stimulated dendritic cells from inducing proliferation of T-cells and visa versa. The ability of the molecule to. vary cytokine secretion could also be tested. In addition direct binding of the candidate molecule to CD36, CD51 or TPS should also be confirmed. This latter confirmation may be easily obtained by applying a sample of the candidate molecule to a purified sample of CD36, CD51 or TPS.
Purified CD36 may be prepared as described by Tandon et al (1989) The Journal of Biological Chemistry, 264 pp 7570-7575. Purified CD51 may be prepared as described by Smith et al, (1990), Journal of Biological Chemistry, 265, 11008-11013 and purified TSP may be prepared as described by Silverstein et al (1985), Journal of Clinical Investigation, 75, pp 2065-2073.
Tests to detect binding of the test molecule are conveniently carried out by immobilizing the CD36, CD51 or TSP to a solid surface, for example the surface of a well of a microtitre plate. Methods of immobilization of protein molecules on such surfaces are well-known to those skilled in the art. The test molecule identified as a CD36 or CD51 agonist is then applied to the immobilized protein. Following removal of unbound test molecule the presence of bound molecule is directly detected. This may be achieved in a number of ways depending on the chemical or biochemical characteristics of the test molecule.
For example where the test molecule is a protein it would be usual to detect binding with a labelled antibody to that protein. If the test molecule is a non-antigenic small molecular weight compound then the compound itself may be radioactively labelled for detection.
The molecule whose activity is to be tested in the method of the invention.may have any type of molecular structure. For example, it may be a protein, a peptide, an amino acid, DNA, RNA, PNA, a nucleotide or a nucleoside, or a low molecular weight compound. It may be a molecule having known pharmacological or biochemical activity or a molecule with no such known activity and may be a novel molecule. The method of the invention is suitable for testing entire libraries of molecules, for example libraries such as would be created by combinatorial chemistry. Indeed, all the embodiments of the - screening method above may be adapted for an automated high throughput compound screen.
Using the method of the invention the present inventors are able to confirm that the Plasmodium falciparum derived protein pf-EMP-1 is an agonist of both CD36 and CD51. In particular a fragment of pf-EMP-1 known as CIDR/A4 which comprises the CD36 binding domain is an agonist of CD36. CIDR/A4 is described by Smith et al (1998) Molecular and Biochemical Parasitology, 97, pp 133-148 and comprises amino acids 402 to 846 of pf-EMP-1 as shown in Figure 2.
Antibodies which bind CD36 and CD51 have also been identified as having agonist activity and are capable of inhibiting the maturation of dendritic cells. Thrombospondin is also an agonist of CD51.
The present invention is also directed to any individual molecule identified as an agonist of CD36 or CD51 by the methods described herein.
The assays of the invention have allowed the inventors to make the further observation that apoptotic cells, the natural ligand of CD36, are also able to inhibit dendritic cell maturation in response to LPS. This is yet further evidence of the role of CD36 in modulating immune response.
In accordance with a second aspect the invention provides a pharmaceutical composition suitable for inducing immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD36 and a pharmacologically acceptable carrier or diluent. The CD36 agonist may be a molecule identified by the method described above. Agonists which are suitable for incorporation into a pharmaceutical composition in accordance with the invention for the treatment of humans include antibodies with an affinity for an epitope of CD36, in particular an antibody which blocks the binding domain on CD36 for pf-EMP-1.
Monoclonal antibodies specific for CD36 which are designated "clone 89" and "clone SM~" and which are commercially available from Serotech or Immunocontact(details above) are suitable for use in the pharmaceutical compositions of the invention.
Other commercially available CD36 antibodies which may be included in pharmaceutical compositions are listed in Appendix 1. It is contemplated that compositions comprising antibodies bispecific against CD36 and CD51 will be useful for inhibiting dendritic cell maturation.
Other agonists suitable for inclusion in pharmaceutical compositions are all variants of the Plasmodum falciparium pf-EMP-1 or fragments of such proteins which comprise the binding domain for CD36.
A particular example is the fragment CIDR/A4 described herein comprising amino acids 402 to 846 of pf-EMP-1.
(Figure 2).
Pharmaceutical compositions comprising a bispecific CD36 antibody and the CIDR/A4 fragment are also contemplated in accordance with the invention.
Yet another agonist suitable for inclusion in a pharmaceutical composition are negatively charged phospholipids such as phosphatidylserine containing liposomes which have also been shown to bind to CD36 and other cellular receptors of immune cells.
Yet another agonist suitable for inclusion in a pharmaceutical composition are apoptotic cells.
In a third of its aspects the invention provides a pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD51 as expressed by mammalian dendritic cells and a pharmacologically acceptable carrier or diluent. As with CD36 acceptable agonists are antibodies, preferably monoclonal antibodies, directed against an epitope of CD51. Particularly suitable are antibodies blocking the binding domain of CD51 for the bridging SUBSTITUTE SHEET (RULE 26) molecule TSP. Antibodies suitable for incorporation in a pharmaceutical composition in accordance with this aspect of the invention are commercially available and set out in Appendix 2.
Thrombospondin (TSP) is also suitable for incorporation into a pharmaceutical composition as a CD51 agonist. Preferably, such compositions also include the Plasmodium falciparum protein pf-EMP-1 or a fragment thereof incorporating the thrombospondin binding domain of pf-EMP-1.
As with CD36, negatively charged phospholipids such as phosphatidylserine are also suitable for incorporation as CD51 agonists in pharmaceutical compositions of the invention as well as apoptotic cells.
Pharmaceutical compositions in accordance with the second and third aspects of the invention are useful for the treatment of autoimmune diseases associated with inappropriate dendritic cell maturation and T-cell proliferation such as systemic lupus erythromatosis, rheumatoid arthritis, autoimmune haemolytic anaemia or idiopathic thrombocytopenic purpura. Vehicles suitable for delivery of pharmaceutically active substances are known to those skilled in the art, especially those for delivery of pharmaceutically active proteins.
In accordance with a fourth aspect of the invention there is provided a method of treating mammalian dendritic cells in vitro to induce immune tolerance therein which comprises exposing said cells to an agonist of cell surface receptors CD36 and/or CD51 as expressed on mammalian dendritic cells. The invention also relates to preparations of cells so treated. Suitable agonists are any of those agonists described above or any molecule or substance identified by the screening method described herein.
Treatment of dendritic cells ex-vivo with an agonist of CD36 and/or CD51 is beneficial in many therapeutic applications as described hereinafter.
For example, in the case of bone marrow transplantation or lymphocyte infusion recipient cells removed from the body are treated with agonists as described above to induce a state of immune tolerance therein. The treated cells are then re-introduced to the body before or simultaneously with the donor cells and the risk of allogeneic reaction is thereby reduced or eliminated. It is contemplated that dendritic cells of the donor may also be treated with a CD36 and/or CD51 agonist to induce immune tolerance. The donor may be allogeneic or xenogeneic.
The present inventors have demonstrated that in mice tolerance to foreign antigens can be achieved by exposure of dendritic cells from one mouse strain, ex-vivo, to a CD51 agonist followed by introduction of the treated cells into another strain of mice. Thus such therapy is expected to be applicable to humans.
In addition the present inventors have shown using a fragment of the a-subunit of the human acetylcholine receptor that immature dendritic cells treated with a CD36 agonist can be "modulated" to induce tolerance against a specific antigen by subsequent ex-vivo exposure to that antigen. Once reintroduced in vivo, immune response to that antigen is reduced or avoided. Thus, dendritic cells may be removed from a patient suffering from an auto-immune disease, for example, and exposed to a CD36 and/or CD51 agonist and an antigenic molecule associated with the auto-immune disease in question and the dendritic cell preparation, with or without maturation, reintroduced into the patient. Alternatively, this method may be used to induce tolerance to a particular allo or xeno-antigen or other therapeutic substance which is likely to induce an unwanted immune response, such as a blood product like factor VIII.
Thus, the invention includes preparations of dendritic cells tailored to the treatment of a particular auto-immune disease by exposure to an agonist of CD36 and/or CD51 and the specific auto-antigen associated with the disease and cell preparations tolerant to other antigens likely to generate an unwanted immune response.
It follows from the inventor's observations concerning inhibition of maturation of dendritic cells with agonists of CD36 and CD51 that a similar effect will be observed with agonists of thrombospondin receptors in general and with other antigen-presenting cells of the immune system which also express CD36 and CD51 and thrombospondin receptors such as macrophages, B-lymphocytes and monocytes.
Thus, in accordance with a fifth aspect the invention provides a method of identifying a molecule which is an agonist of cell surface receptors CD36 and/or CD51 and/or a thrombospondin receptor as expressed on antigen-presenting cells of the mammalian immune system which method comprises:
a) exposing mammalian antigen-presenting cells to the molecule to be tested, b) exposing said cells to an immune stimulus and c) determining the response to said immune stimulus by said cells, wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule under test is a CD36 and/or CD51 agonist or an agonist of a thrombospondin receptor.
Preferably, the response that is measured is maturation of said antigen presenting cell. Such a screening method may be carried out using the general methodology already described herein for dendritic cells. For example, monocytes can be purified from peripheral blood by adherence of PBMC to plastic dishes. Non-adherent cells are removed and the adherent cells can be detached by incubation with EDTA
in PBS. Contaminating lymphocytes are depleted with the aid of magnetic heads and antiCD3 and antiCDl9 monoclonal antibodies. Macrophages may be generated by culturing monocytes, which have been isolated as described above, in RPMI 1640 supplemented with M-CSF
for six days. ~i-lyphocytes can be isolated from blood by virtue of their non-adherence to plastic petri dishes. The non-adherent cells are subjected to depletion of contaminating monocytes and T-cells by exposure to magnetic heads and antiCDl4 and antiCD3 monoclonal antibodies.
Once isolated the antigen presenting cells are exposed to a substance to be tested for agonist activity against CD36, CD51 or a thrombospondin receptor and the degree of activation of said cells is measured. As with dendritic cells, activation may be determined by measuring the levels of secretion of various cytokines, or by testing ability of said antigen presenting cells to stimulate T-cell proliferation. In a preferred embodiment the increased expression of certain cell surface receptors is used as a measure of activation. In the case of monocytes and macrophages activation is accompanied by an increase in surface expression of HLA-DR, CD54 and CD86 which is measured in the manner described above, preferably with the use of a monoclonal antibodies to HLA-DR, CD54 and CD86. B-cell activation is determined by measuring the level of cell surface expression of HLA-DR, CD86 and CD40. The expression may be detected using antibodies to these cell surface receptors. Such as clone BF1, Serotech (HLA DR) clone BU63, Serotech (CD86) and clone TRAP1 (Pharmingen) (CD40) .
The invention also further relates to uses of an agonist as identified above using said antigen-s presenting cells for treatment of any of the auto-immune diseases listed above and for inducing immune tolerance in said antigen presenting cells ex-vivo as well as to antigen-presenting cell preparations which have been treated with a CD36 and/or CD51 agonist and/or thrombospondin receptor agonist and optionally an antigenic material.
The invention also relates to pharmaceutical compositions comprising an agonist of a thrombospondin receptor, for example a"~33 or a~~35, with a pharmaceutically acceptable carrier or diluent suitable agonists include antibodies to the thrombospondin binding domain of said receptor, for example any of the antibodies listed in Appendix 3.
Other suitable agonists include negatively charged phospholipids such as phosphatidylserine containing liposomes.
It is a further conclusion from the work of the present inventors that agonists of (3-integrin associated with the cell surface receptor CD51 as expressed on the surface of antigen-presenting cells of the mammalian immune system, will also be useful for inducing immune tolerance. The invention in a sixth aspect thus, further relates to methods of identifying ~3-integrin agonists by any of the procedures described above and to uses of ~i-integrin agonists, as defined above, for any of the medical uses which are described herein.
In a seventh aspect the present invention further relates to uses of apoptotic cells as a medicament for inducing immune tolerance in antigen-presenting cells, preferably dendritic cells and to pharmaceutical compositions comprising those cells in a suitable carrier or diluent. Apoptotic cells are suitable for delivering tissue specific antigens including major and minor histocompatibility antigens to dendritic or other antigen-presenting cells. Delivering antigens in this way allows delivery of unknown antigens or antigens where the class II restricted epitope(s) are not defined. The tissue origin of the apoptotic cell may be varied depending upon the application. For example, it is preferred for the apoptotic cell to be of the same tissue type as any cell bearing an antigen to which tolerance is to be induced.
In accordance with an eighth aspect the invention further relates to the use of negatively charged phospholipids for inducing immune tolerance in antigen presenting cells. Said immune tolerance may be induced by treatment of said antigen presenting cells, for example dendritic cells, with said negatively-charged phospholipid either ex-vivo by the methods described herein or by administration of the phospholipid to a patient by any of the conventional administration routes known to those skilled in the art. A preferred form of composition is liposomes comprising the negatively charged phospholipid. A
preferred phospholipid is phosphatidylserine.
Since the inventions of the present application were developed following the basic observation that Plasmodium falciparum infected erythrocytes adhere to dendritic cells and inhibit the maturation thereof it follows that molecules which block or inhibit such adherence may be useful as pharmaceuticals in the clinical management of malaria, in particular molecules which inhibit adherence of parasite-infected erythrocytes to CD36 or TSP.
Thus, in accordance with a ninth aspect of the invention a method comprising the following steps is used to identify a molecule capable of preventing adherence of erythrocytes infected with a malarial parasite to human dendritic cells:
(a) exposing a purified preparation of CD36 or TSP to:-(i) the molecule~to be tested and (ii) parasitsed human erythrocytes either consecutively or simultaneously and (b) determining the level of adherence of said parasitised erythrocytes to said CD36 or TSP
wherein a reduction in the level of adherence to CD36 or TSP in the presence of the test molecule compared to the level of adherence in the absence of said test molecule is an indication that said molecule is capable of preventing the adherence of erythrocytes infected with the malarial parasite to human dendritic cells.
The erythrocytes may be infected with Plasmodium falciparum or another Plasmodium species. Suitable falciparum strains include ITO/A4 or ITO/C24 which may be derived as described by Roberts et al (1992) Nature 357 pp 689-692 or Malayan Camp (MC) which may be obtained as described by Roberts et al (1985) Nature 318:64-66.
A suitable format for carrying out a screening method as described above is to immobilize the purified CD36 or TSP onto a solid surface.
Preferably, immobilization is secured by adsorption of the protein molecules to a plastic surface such as a petri dish. Parasitised erythrocytes suspended in a suitable binding medium are added to the adsorbed CD36 or TSP and incubated for a period sufficient to allow adherence, for example, about 1 hour. Thereafter the binding medium and any non-adhered erythrocytes are removed and a suitable erythrocyte stain for example, Giemsa, added to the petri dish. Adhered erythrocytes may be quantified by counting under a light microscope. Alternatively, depending on the stain used, erythrocyte adherence may be quantified by spectrometry, fluorescence microcopy and the like.
In a tenth aspect the invention provides a method of identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells which comprises:
a) exposing immature human dendritic cells to the Plasmodium falciparum protein pf-EMP-1 or an active binding domain thereof in the presence or absence of the molecule to be tested, b) exposing said immature dendritic cells to an immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, wherein any maturation of said dendritic cells in the presence of the test molecule over and above that manifested in the absence of said molecule is an indication that said molecule is capable of preventing adherence of red blood cells infected with a malarial parasite to human dendritic cells.
Maturation of dendritic cells may be measured by any of the methods already described herein. Suitable immune stimulants include LPS, TNFa, CD40L and monocyte conditioned medium (MCM). Preferably the pf-EMP-1 preparation for use in the method is that designated in pf-EMP-1 A4var as described by Smith et al (see before) and having the Genbank Accession No.
L42244. The fragment CIDR/A4 may also be used.
In a further aspect the invention provides for use of molecules identified by the aforementioned methods which inhibit infected erythrocyte adherence to dendritic cells in pharmaceutical compositions for the treatment of malarial infection.
Based on the present inventors' observations it is further contemplated that a modified CIDR region of the pf-EMP-1 A4 variant protein could be incorporated in a multisubunit vaccine against falciparum malaria.
This would induce blocking antibodies against the CD36 binding domain of pf-EMP-1 variant proteins so that the immune responses against other proteins are not inhibited.
All documents cited in this application are incorporated herein by reference.
The invention will now be further described with reference to the following Figures and Examples.
FIGURE 1 shows schematically the molecular basis for the binding of Plasmodium falciparum infected red blood cells to CD36 and TSP on the surface of dendritic cells;
FIGURE 2 shows the amino acid sequence of the pf-EMP-1 fragment CIDR/A4;
FIGURE 3 shows the increase in surface expression of dendritic cell marker antigens HLA DR, CD54, CD40, CD80, CD83 and CD86 following immune stimulation after exposure to (a) LPS matured dendritic cells, (b) dendritic cells matured with LPS, with and without prior exposure to RBC, (c) dendritic cells matured with LPS with and without prior exposure to parasite lysate and (d) dendritic cells matured with LPS with and without prior exposure to intact ITO/A4 infected RBC;
FIGURE 4; (A) shows the absolute binding of erythrocytes infected with parasite lines ITO/A4, ITO/C24, MC and T9/96 to CD54, CD56, and TSP (a,c,e,g) and (B) shows the increase in surface expression of LPS matured dendritic cells compared with dendritic cells exposed to the respective parasite line prior to maturation (b,d,f,h);
FIGURE 5 shows transmission electron micrographs illustrating the interaction of dendritic cells with (a) ITO/A4 infected erythrocytes and (d) non-adherent T9/96 infected erythrocytes;
FIGURE 6 shows dendritic cell stimulation of T-cell proliferation (a) induced by immature dendritic cells (~), LPS-matured dendritic cells (~) and dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (~) prior to maturation, primary CD4+ T-cell responses to parasite lysate (b) and to keyhole limpit haemocyanin (c) induced by LPS-matured autologous dendritic cells (D, o) and autologous dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (~,~)prior to maturation;
FIGURE 7 shows the effect of monoclonal antibodies against CD36 and CD51 on maturation of dendritic cells represented graphically as relative increase in surface expression of dendritic cells matured with LPS
compared with immature dendritic cells;
FIGURE 8 shows the effect of monoclonal antibodies against CD36 and CD51 on dendritic cell maturation as a FACscan output;
FIGURE 9 shows further results of experiments with apoptotic cells (a) output of FACscan,(b) staining with potassium iodide to exclude dead cells, (c) proliferation of allogenic T-cells stimulated by increasing numbers of immature dendritic cells,(~)LPS-matured dendritic cells (1) or dendritic cells exposed to apoptotic dendritic cells and then matured with LPS(~).
FIGURE 10 shows the effect of apoptotic neutrophils on the maturation of dendritic cells;
FIGURE 11 shows results bf a T-cell proliferation assay including antigen specific T-cell proliferation.
(a) Proliferation of allogeneic T-cells. (b) proliferation of KLH specific CD4+CD45R0- autologous T-cells (c, d) proliferation of the T-cell clone TB-2 specific for the human Acetylcholine Receptor a-subunit in response to polypeptide (c) or peptide (d).
Stimulator dendritic cells were treated as follows:
immature DC alone (") or matured with LPS (0);
dendritic cells exposed to irrelevant antibodies with (O) or without (~) antigen and then matured with LPS;
dendritic cells exposed to antiCD36 antibody with (v) or without (~) antigen and then matured with LPS;
dendritic cells exposed to antiCD51 antibody with (o) or without (1)antigen and then matured with LPS;
dendritic cells exposed to antiCD36 and antiCD51 antibody with (p) or without (~) antigen and then matured with LPS.
FIGURE 12 shows secretion of cytokines TNFa, IL12p70 and IL10 by dendritic cells exposed to an antiCD36 antibody or to apoptotic dendritic cells and respective controls;
FIGURE 13 shows in vitro maturation of mouse dendritic cells following exposure to an antiCD51 antibody; and FIGURE 14 shows results from mouse popliteal lymph node assay.

Generation of antigen-presenting cells (a) Dendritic cells Immature dendritic cells were derived from peripheral human blood cells using standard procedures as described by Sallusto et al (1995) J. Exp. Med. 182 pp 389-400. Briefly, monocytes were cultivated in RPMI 1640 supplemented with 2mM Glutamine,50 pg/ml Kanamycin, to nonessential amino acids (GibcoBRL), l00 human AB serum and 50 ng/ml of each IL-4 (specific activity >2x106 U/mg, PeproTech) and GM-CSF (specific activity > 1x10' U/mg, Schering-Plough) for 6 days.
Between day six and day nine of the culture non-adherent immature dendritic cells were harvested and purified by depletion of contaminating lymphocytes with the aid of magnetic beads (Dynal) and anti-CD3 and anti-CD19 monoclonal antibodies (DAKO).
(b) Monocytes Monocytes were purified from peripheral blood by adherence of PBMC to plastic dishes for 2 hours. Non adherent cells were removed and the adherent cells layer washed 2 times with warm PBS. For further purification, the adherent cells were detached by incubation with 2 mM EDTA in PBS for 20 min and contaminating lymphocytes depleted with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD3 and anti-CD19 monoclonal antibodies (DAKO).
(c) Macrophages Monocytes isolated as described above were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 ug/ ml Kanamycin, 10% human AB serum and 50 ng/ml of M-CSF
(specific activity > 2 x 106 U/mg, Peprotech) for 6 days.
(d) B-lymphocytes B-lymphocytes were isolated from human blood according to standard procedures. Briefly, non-adherent PBMC
were subjected to depletion of contaminating monocytes and T-cells with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD14 and anti-CD3 monoclonal antibodies (DAKO). B-cells were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 ug/ ml Kanamycin, to to loo human AB serum.
(e) CD34+ Cells CD34+ cells were isolated from PBMC with the aid of anti-CD34 antibody conjugated magnetic beads (Dynal or Miltenyi). CD34+ progenitor were then cultured,in RPMI
1640 supplemented with 2 mM Glutamine, 50 ug/ ml Kanamycin, to to lOs human AB serum and the following cytokines: 100 ng/ml of GM-CSF (Schering-Plough), 50 ng/ml TNFa and 50 ng/ml SCF (Peprotech) for 12 days.
As an alternative, CD34+ cells could be expanded in the above mentioned medium but supplemented with 25 ng/ml FLT3-L , l0U/ml TPO, SCF 20 ng/ml (Peprotech) for up to 8 weeks and then induced to differentiate to dendritic cell by culture of a further 3 days in medium supplemented with 25 ng/ml GM-CSF and 25 ng/ml IL-4."

Maturation assay (a) For maturation assays 1x106 purified dendritic cells were incubated in duplicate wells (a) with 100 ng/ml LPS,(b) with 100ng/ml LPS with or without prior exposure to 1x10$ RBC, (c) with 100 ng/ml LPS
with or without prior exposure to parasite lysate corresponding to 1x108 parasite infected RBC,(d) 100 ng/ml LPS with or without prior exposure to 1x108 intact ITO/A4 infected RBCs. Incubation with LPS
(Salmonella typhimurium) was for a period of 48 hours.
Maturation of the dendritic cells was measured using monoclonal antibodies to the following human cell surface markers: CD3 clone OKT3, HLA A,B,C clone W32/6, CD14 clone Tuk4, CD54 clone 6.5B5, CD19 clone HD37 (DAKO): CD36 clone 89 (IgGl) or clone SMQ(IgM), CD80 clone BB1, CD40 clone LOB7/6, CD86 clone BU63, HLA DR clone B-F-1 (Serotec), CD83 clone HBl5a (Zhou et al (1995) J. Imm. 154, pp3821-3835. Staining of dendritic cells was performed as described by Zhou et al above and immunofluorescence analysed by FACScan (Becton Dickenson). All experiments were repeated at least six times with dendritic cells obtained from different donors. Dead cells were excluded from analysis using Propidium Iodide. The results are shown on Figure 3. The relative increase of surface expression is expressed as the mean fluorescence intensity (MFI) of matured dendritic cells over the MFI on immature dendritic cells.
The results show that dendritic cell maturation is inhibited by the direct interaction with intact infected erythrocytes and is not due to the secretion of inhibitory parasite products or a toxic effect of parasite debris.
The differences in surface expression on dendritic cells exposed to intact infected erythrocytes to dendritic cells alone are statistically significant for all markers with p<0.01 (Student t-test).

(b) Other antigen-presenting cells For maturation assays 1 x 106 purified monocytes or macrophages were incubated in duplicate wells with or without LPS in the presence of medium alone, antibodies such as anti-CD36 or anti-CD51 or control antibodies, apoptotic cells or necrotic cells.
Maturation was measured by the increase in surface expression of HLA-DR, CD54 and CD86 using the antibodies and flow cytometry as described above.
For maturation assays 1 x 106 purified B-cells were incubated in duplicate wells with or without LPS in the presence of medium alone, antibodies such as anti-CD36 or anti CD51 or control antibodies, apoptotic cells or necrotic cells. Activation was measured by the increase in surface expression of HLA DR (clone BF-1, Serotec), CD86 (clone BU63, Serotec) and CD40-Ligand (clone TRAP1, Pharmingen) and flow cytometry.
EXAMPhE 3 Cultivation of Plasmodium falciparum infected red blood cells Laboratory strains of Plasmodium falciparum were cultured in human RBC as described by Trager et al (1976) Science. 193 pp673 to 675. The cytoadherent cell lines ITO/A4 and ITO/C24 were clones isolated by manipulation from the IT04 line, which is derived from a parasite isolate from Ituxi in Brazil. The cytoadherent parasite line Malayan Camp (MC) and the non-adherent cell line T9/96 were both adapted to in vitro culture from parasites originally isolated from Thailand., All cultures were free from mycoplasma contamination. Infected erythrocytes were purified either by differential sedimentation in Plasmagel or through 65% Percoll both of which gave a yield of more than 90% infected erythrocytes. Examination of a thin film revealed that more than 90% of infected erythrocyes were viable. Parasite lysate was obtained by three rounds of freezing and thawing of mature infected RBC. Parasite pigment was prepared as described by Schwarzer et al (1994) BR. J. Haematol.
88, pp740-745. Parasite conditioned medium was the supernatant derived after culturing 1x108 purified infected erythrocytes in dendritic cell medium for 24 hours. All materials were from Sigma unless otherwise stated.

Binding of parasites to purified proteins Binding of parasitised RBCs to purified proteins was measured as previously described by Craig et al (1997) Infect. Immun. 65, pp 4580-4585. Briefly, two microlitres of a solution of TSP (Gibco-BRL), purified CD36 or purified CD54 (ICAM-Fc) were adsorbed onto bacteriological, plastic plates. Mature erythrocytes parasitised with P. falciparum strains (a) ITO/A4, (c) ITO/C24, (e) MC and (g) T9/96, were suspended in binding medium and added to each dish. The erythrocytes were allowed to settle and then resuspended by gentle rotation every 10 minutes for 1 hour. Non-adherent cells were removed, the remaining cells fixed and stained with Giemsa. Adherent parasitised cells were counted by light microscopy and the number of cells bound per square millimeter were corrected to binding at 2o haematocrit and 5°s parasitaemia. The results are shown in Figure 4A and confirm that like ITO/A4, ITO/C24 and MC are able to adhere to CD36 and TSP. However, their adherence to CD54 was much reduced. T6/96 does not adhere to CD54, CD36 or TSP.

Effect of parasite strains on maturation A maturation assay as described in Example 2 was carried out but exposing immature dendritic cells to erythrocytes infected with (b) ITO/A4, (d) ITO/C24, (f) MC and (h) T9/96. The results are shown in Figure 4B. While parasite lines MC and ITO/C24 inhibited the maturation of dendritic cells in a similar vein to clone ITO/A4, the non-adherent line T9/96 did not inhibit maturation of dendritic cells even at a ratio of infected erythrocytes to dendritic cells of 100:1.

Electron microscopy Adherence of ITO/A4 infected erythrocytes but not T9/96 infected erythrocytes to dendritic cells was confirmed by electron microscopy. One million purified immature dendritic cells were incubated for 2 hours and for 12 hours with 1x108 ITO/A4 infected RBC
(a) or T9/96 infected (d) in 2 ml of dendritic cell medium , harvested and fixed with 2.5%
glutaraldehyde/cacodylate buffer. Cells were post fixed in osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate prior to examination in a Joel 1200EX electron microscope. The number of adherent and infected erythrocytes and the number of phagosomes containing pigment granules.was counted in each sample in thin sections of 100 randomly selected dendritic cells. Transmission electron micrographs are shown in Figure 5.
Note the cell processes partially enclosing infected erythrocytes (arrows in a) and the close apposition of the limiting membranes of the infected erythrocytes and dendritic cells particularly at the knobs (b, arrowhead). Within dendritic cell cytoplasm are phagosomes containing characteristic pigment granules (c, arrows). N -dendritic cell nucleus, P - infected erythrocyte. Bars are 2 ~m (a and d), 200 um (b), 500 um (c) .
ITO/A4 infected erythrocytes were observed to be in intimate contact with immature dendritic cells with cytoplasmic processes partially enclosing the parasites (Fig. 5a). The plasmalemma of the infected erythrocytes was in close apposition to the limiting membrane of the dendritic cell particularly at the site of knobs (Fig. 5b). A similar apposition between parasitised erythrocytes and host cells is seen between infected red blood cells and endothelial cells (Berendt et al (1994) Parasitology 108 Suppl. 519-28).
In contrast, only a few infected erythrocytes of the T9/96 strain were associated with the dendritic cells (Fig. 5d). When quantified, ten times more ITO/A4 infected erythrocytes were found adherent to dendritic cells than T9/96 infected erythrocytes in 100 thin sections of dendritic cells. Furthermore, ingestion of intact ITO/A4 infected erythrocytes by dendritic cells was not observed during this time. Nevertheless, phagocytosis of parasite debris as revealed by the number of phagosomes containing pigment granules (Fig.
5c) was similar for dendritic cells incubated with ITO/A4 or with T9/96.

T-cell proliferation assays Total-T-cells (allogeneic MLR) or CD4+ T cells (primary T-cell responses) were purified using a Cellect column (TCS). For the allogeneic MLR, dendritic cells were added in increasing numbers (156 to 10,000) to 1 x 105 T-cells in triplicate and incubated for 5 days. T-cells were pulsed with 0.5 uCi 3H-thymidine/well for the last 18 hours of the culture. For primary T-cell responses, 1 x 106 dendritic cells were incubated with medium alone or with 1 x 10$ infected erythrocytes for 18 h and then pulsed with 10 ug/ml parasite-lysate or with 30 ug/ml keyhole limpet haemocyanin, respectively. The dendritic cells were purified by sedimentation through LymphoprepTM and 1 X 105 dendritic cells were culterd with 1.5 x 106 CD4+ T-cells from the same donor. From day 4 to day 6 of culture, 50 u1 aliquots were taken in triplicate and pulsed with 0.5 uCi 3H-thymidine/well for 8 hours.
(see Plebanski et al (1992) Immunol. 75 86-90). The results are shown in Figure 6.
Dendritic cells exposed to intact infected erythrocytes are poor stimulators of T-cell proliferation. Allogeneic T-cell proliferation (a) induced by immature dendritic cells (~), LPS-matured dendritic cells (~) and dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (1) prior to maturation. Primary CD4+ T-cell responses to parasite-lysate (b) and to keyhole limpet haemocyanin (c) induced by LPS-matured autologous dendritic cells (O, o) and autologous dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (~,~) prior to maturation. Data from one out of three independent experiments are shown.
Dendritic cells matured after incubation with uninfected RBC, a crude pigment preparation or a lysate of infected erythrocytes induced a similar degree of T-cell proliferation in a mixed leukocyte, reaction to that induced by control mature dendritic cells (data not shown).
However, dendritic cells incubated with LPS after exposure to intact infected erythrocytes from the parasite line ITO/A4 were strikingly less efficient in their induction of T-cell proliferation compared with the T-cell proliferation induced by mature dendritic cells (Fig 6a). Furthermore, dendritic cells exposed to intact infected erythrocytes before maturation with LPS did not induce primary CD4+ T-cell responses to lysate of infected erythrocytes or to keyhole limpet haemocyanin (Plebanski et al) (Fig 6, b,c).
It is concluded that the maturation of dendritic cells and their subsequent ability to activate T-cells is profoundly inhibited by their interaction with intact infected erythrocytes. Non-adherent parasite lines, parasite debris and crude pigment do not modulate dendritic cell function in this way.
These studies provide one explanation for the clinical and experimental evidence of immune dysregulation during malaria infection such as the impairment of the delayed-type hypersensitivity response to recall antigens and the antibody response to vaccines. -Maturation assay with monoclonal antibody A maturation assay was carried out as described in Example 2 except that instead of infected erythrocytes the immature dendritic cells were exposed to monoclonal antibodies to CD36, CD51 or both prior to immune stimulation with LPS.
Specifically, 1x106 purified dendritic cells were incubated in duplicate wells without or with either 25 ug irrelevant IgM antibody, 25 ~Zg irrelevant IgGl antibody, 25 ug antiCD36 antibody, 25 ug antiCD51 antibody or a combination thereof for at least 3 hours. Thereafter, dendritic cells were matured with 100 ng/ml LPS (Salmonella typhimurium) for 48 hours or left untreated as a control. The monoclonal antibodies tested were CD36 clone SMQ (Immunocontact) and clone 89 (Serotec), CD51 clone 13C2 (Immunocontact), IgM isotope control clone MOPC, IgG
isotope control clone MOPC (Sigma). The results of two experiments are shown in Figures 7 and 8 respectively. As will be apparent both CD36 and CD51 antibodies have the effect of inhibiting dendritic cell maturation in a similar manner to infected erythrocytes.
EXAMPhE 9 Maturation assay with apoptotic cells A maturation assay was carried out as described in Example 2 except that instead of infected erythrocytes the immature dendritic cells were exposed to apoptotic cells prior to immune stimulation with LPS. Apoptotic or necrotic cells were derived from purified autologous dendritic cells, monocytes or neutrophils. Specifically for maturation assays in the presence of apoptotic bodies 1x106 purified dendritic cells were incubated in duplicate wells without or with either 2 x 106 autologous apoptotic or necrotic cells for 12 hours.
Maturation was induced by the addition of LPS or TNFa as stated above. Apoptosis was induced by radiation with a calibrated UV lamp at a dose of 2500 mJ/cmz and evaluated by staining with FITC-AnnexinV/Propidium Iodide according to manufacturers recommendations (Roche Diagnostics) 3 hours and 12 hours after UV
radiation. Necrosis was induced by at least three cycles of rapid freezing at -70°C and thawing at 37°C.
Thereafter, more then 900 of cells were permeable for trypan blue. The results are shown in Figure 9 as follows:
Apoptotic cells but not necrotic cells inhibit the maturation of dendritic cells. (a) Immature dendritic cells were left untreated, matured with LPS or exposed autologous apoptotic or necrotic dendritic cells prior to maturation with LPS and subsequently stained with antibodies directed against surface marker and analysed by FACScan as indicated. (b) Dead Cells and especially apoptotic cells were efficiently excluded from analysis by gating on forward scatter and exclusion of cells positive for Propidium Iodide.
(c) Proliferation of allogeneic T-cells stimulated by increasing numbers of immature dendritic cells (~), LPS-matured cells (1) or dendritic cells exposed to apopotic dendritic cells and matured with LPS (~).
The results of a further experiment with apoptotic neutrophils shown in Figure 10.

Antigen specific T-cell responses The proliferative response of the CD4+ T-cell clone TB-2 for the peptide 144-163 of the human acetylcholine receptor (Nagvekar N et al, J. Clin in est, 1998 101 (10) pp 2268-77) was analysed. T-cell proliferation was measured as described in Example 7.
For antigen-specific T-cell responses, 1 x 106 dendritic cells were incubated with medium alone or with antibodies as indicated and then pulsed for 6 h with 0.025 mM AChR a: 3-181 polypeptide before or 1 mM AChR a:144-163 peptide after maturation with LPS.
For antigen specific T-cell responses of the clone TB-2 increasing numbers of MHC class II matched dendritic cells were incubated with 3 x 104 T-cells for 72 h. Proliferation was measured in all assays by adding 0.5 mCi 3H-thymidine/well for the last 8 hours of the culture.
The results are shown in Figure 11 (c) and (d).
Exposure of dendritic cells to antiCD36, antiCD51 or both antibodies abolished their ability to induce proliferation in the T-cell clone as compared to dendritic cells exposed to irrelevant antibodies (Fig 2c, d). The proliferation of the T-cell clone remained low even when the modulated dendritic cells were exogenously loaded with peptide thus excluding a defect in antigen-uptake or antigen-processing due to the presence of antibodies.

Cytokine production by dendritic cells Secretion of the cytokines TNF alpha, IL12 p70 and IL10 was measured in supernatants of dendritic cells treated with either anti-CD36 antibody or apoptotic cells or in the respective controls before or after maturation with LPS for 24 hours using commercially available ELISA kits. These kits can be obtained from R and D Systems, Europe Ltd. 4-10, The Quadrant, Barton Lane, Abingdon, Oxford, OX143 YS and BD
Pharmingen, 10975, Torreyana Road, San Diego, CA
92121, USA.
Maturation with LPS induced secretion of TNF-a by dendritic cells irrespective whether they were exposed to anti-CD36 antibodies or apoptotic cells although the concentration of TNF-a was consistently slightly lower than in the respective controls. IL12 p70 was secreted by control dendritic cells matured with LPS whereas IL10 was secreted by dendritic cells exposed to anti-CD36 antibodies or apoptotic cells. Of note, the absolute amount of IL10 varied considerably between dendritic cells treated with anti-CD36 and dendritic cells exposed to apoptotic cells in response to LPS. It is possible that intact cells bind to more than one receptor thus modifying the cytokine secretion induced by CD36 alone. However, we investigated whether secretion of IL10 had a role in inhibition of dendritic cell maturation due to ligation of CD36 by maturing dendritic cells exposed anti-CD36 antibodies or to apoptotic cells in the presence of blocking anti-IL10 antibodies. The inhibition of dendritic cell maturation was not reversed (data not shown) and is therefore independent of the secretion of IL10.

Modulation of mouse dendritic cell maturation and function by antiCD51 antibodies in vitro and in vivo Rationale Since human monocyte derived dendritic cells can be modulated in their maturation and function by a variety of agents including antibodies binding to CD36 and or CD51, in this study we began to investigate whether a similar phenomenon could be observed in mouse dendritic cells.
Methods Generation of bone-marrow derived dendritic cells: Bone marrow from male Balb/c (H-2k ) mice was harvested and total cells were cultured in RPMI supplemented with 2 mM glutamine, 50 mg Kanamycin, 10 % FCS,10 ng/ml each murine recombinant GM-CSF and IL-4. On day two of culture half the medium was replaced with fresh medium supplemented with cytokines and on day four of culture non-adherent cells were harvested.
In vitro maturation of bone-marrow derived mouse dendritic cells: One million of bone marrow derived dendritic cells (approximately 50% total cells) in duplicate were exposed to either medium alone, 25 mg isotpype control antibody or 25 mg antiCD51 antibody for 8 hours. Cells were subsequently exposed to 100 ng LPS for 48 hours or left alone as a control. Maturation of dendritic cells was analysed by double staining with FITC conjugated antibodies against CDllc and PE-conjugated antibodies directed against either CD40, CD54, CD86 or I-A and subsequent FACScan analysis.
Analysis was performed on CDllc-FITC positive cells.
Popliteal lymph node assay: Dendritic cells were exposed to medium alone or to antiCD51 antibodies and then matured with LPS as described above. The cells were then harvested and washed four times in PBS in order to remove LPS. Cells were resuspended in 10%
FCS/PBS at a concentration of 6 x 105 total cells/20 ml.
Groups of six male C3H3/HE (H-2d) mice were injected with 20 ml of PBS into the right footpad and LPS
matured dendric cells into the left footpad, with 20 ml of PBS into the right footpath and dendritic cells exposed to antiCD51 antibody prior to LPS maturation into the left footpad or with 20 ml of PBS into the right and the left footpad. After one week mice were sacrificed and the popliteal lymphnodes were removed.
The weight of the left and the right lymphnode of each mouse in all three groups were determined and the ratio of the weight of the left lymphnode to the weight of the right lymphnode was calculated. The mean and SE of the ratio was determined for each group.
Results Dendritic cells matured with LPS (DC LPS) increased the surface expression of the molecules CD40, CD54 and CD86 as compared to immature dendritic cells (DC). However, when dendritic cells were treated with antiCD51 antibodies prior to exposure to LPS (DC CD51 lps), the dendritic cells failed to mature and the expression of surface molecules remained at the level of immature dendritic cells (DC). The antibody itself had no effect on dendritic cell maturation (DC CD51). The results are shown in Figure 13.
When dendritic cells matured with LPS were injected into the footpad of allogeneic C3H/HE mice, they induced a strong allogeneic T-cell response as measured by the increase in weight of the popliteal lymphnode of the right footpad compared to the popliteal lymphnode of the left footpad. By contrast, the increase in weight of the right popliteal lymphnode over that of the left popliteal lymphnode was markedly reduced when dendritic cells were exposed to antiCD51 antibody prior to maturation with LPS. However, the overall increase in weight was still above that of popliteal lympnodes of mice that had been injected with PBS in both footpads. The results are shown in Figure 14.

EXAMPhE 13 Modulation of human dendritic cells ex vivo Agonists of CD36, CD51, thrombospondin receptors or ~i-integrin may be used to modulate human immune response in patients with unwanted and/or harmful allo- or auto-immune responses. For such a method of treatment immature human dendritic cells which are defined and identifiable as described herein and with the ability to phagocytose are derived from preparations of human peripheral blood.
Specifically, the dendritic cells are derived from CD34+ stem cells or monocytes isolated from human peripheral blood by the method described in Example 1. To the preparation of cells suspended in appropriate medium, for example RPMI as defined herein, an agonist of C36, CD51, thrombospondin receptor or (3-integrin is added. The relative concentration of agonist to cells is adjusted depending on the nature of the agonist used. For example, if the agonist is a monoclonal antibody, about 25 ug antibody to about 106 dendritic cells is appropriate at a concentration of 25 ug M1-1' The cells are treated for between 3 and 24 hours.
Depending on the particular application cells are returned to the individual from which they were originally derived or administered to.another individual. Adminitration may be by intravenous infusion, by inhalation or by sub-cutaneous or intramuscular injection.
Administration of dendritic cells to a human following the method described above will give rise to a generalized immune suppressive effect which will be useful, in a number of situations, for example in the prevention of rejection of allografts and xenografts or for treatment of disease suspected of having an auto-immune basis but for which the auto-antigen is not known.
However, the method described above may be modified to produce dendritic cells which are tolerant to a specific antigen. In this case the dendritic cell preparation is exposed to an antigen against which tolerance is to be induced as well as to the CD36, CD51, thrombospondrin receptor or (3-integrin agonist. The cells may be exposed to antigenic material, before, after or simultaneously with the aforesaid agonist molecule. The antigenic material may be linked to, fused to or otherwise associated with said agonist molecule. Exposure to the antigenic material is for about 6 to about 24 hours with or without an immune stimulant, then the cells are reintroduced to the patient as described above.
The ability to induce tolerance to a specific antigen, for example an allo, xeno or auto-antigen allows a great many therapeutic applications. For example tolerance can be induced in respect of the following antigens:
to major or minor histocompatibility antigens of a recipient of a bone marrow transplant (to modulate graft versus host disease in bone marrow transplantation in dendritic cells from the bone marrow donor) or to donor major or minor histocompatibility antigens in dendritic cells of recipients of solid organ transplant or to antigens to which there is pathological immune response causing auto-immune diseases for example:
components of autologous red blood cells to modulate the immune response in patients with auto-immune hemolytic anaemia components of autologous platelets in patients with auto-immune thrombocytopeniacomponents of beta islet cells of the pancreas in patients with insulin dependent diabetes mellitus components of other endocrine organs in patients with other organ specific auto-immune diseases components of the acetylcholine receptor in patients with myasthenia gravis other antigens or apoptotic cells containing antigens causing harmful or pathological immune responses in other auto-immune diseases to antigens to which there is pathological immune response causing atopic or allergic diseases, for example antigens eliciting an immune response in hay fever, asthma, eczema or coeliac disease to antigens to which there is pathological immune response that may be defined in other diseases for example in non-organ specific immune diseases (systemic lupus erythematosis or rheumatoid arthritis) or other immune mediated arthritis or other connective tissue diseases in inflammatory bowel disease in auto-immune hepatitis in multiple sclerosis or in other auto-immune disease to alto-antigens to which there is a harmful or pathological immune response for example components of red blood cells in haemolytic disease of the newborn or in previously transfused patients components of platelets for example in neo-natal allo immune thrombocytopenia or in conditions where there is an allo-immune response to transfused platelets other blood products or substitutes for example Factor VIII in haemophilia patients other synthesized or manufactured or naturally occurring products or substances Although the treatment of humans with dendritic cells is described in this example it will be appreciated that other antigen-presenting cells, such as macrophages, monocytes or (3-lymphocytes could be used for treatment with an agonist of CD36, CD51, thrombospondin receptor or (3-integrin and optionally an antigenic material. Specific tolerance can be introduced in such cells for use in any of the applications listed above.
Example 14 Preparation of phosphatidylser.ine liposome compositions and their therapeutic uses.
Liposomes containing phosphatidylserine or other negatively charged phospholipids, with or without additional.targeting molecules, induce general immune unresponsiveness. Liposomes encapsulating antigens and phospholipids with or without additional targeting molecules induce antigen specific immune unresponsiveness.
Liposomes are prepared as described by Coradini et al, Anticancer Research 1998 18 177-182. In brief clean glass tubes are coated with 2 micromolar of mixtures of phosphatidylcholine and phosphatidylserine, other negatively charged phospholipids or other phospholipids including cholesterol and/or cholesterol ester dissolved in chloroform. The solvent is evaporated under nitrogen gas and the tubes incubated in a vacuum for 45 minutes. Sterilised phosphate buffered saline (unmodified liposomes) or containing the antigens) to which unresponsiveness will be induced with or without molecules allowing targeting of the liposomes to CD36 and/or CD51 and/or beta-integrins and/or other receptors of apoptotic cells or other molecules expressed on the surface of antigen presenting cells, is added to the lipid shell. Suitable targeting molecules are monoclonal antibodies to the respective receptors or fragments of the P. falciparum erythrocyte membrane protein-1 that bound to CD36 and/or thrombospondin. The tubes are shaken at high' speed for 5 minutes and separated from free fatty acid by ultracentrifugation at 100,0008 for 60 minutes. Targeting molecules (see above) may be covalently or non-covalently attached to the surface of liposomes. The liposomes are filtered through a 0.22 micrometer filter. Encapsulation of antigens and targeting molecules can also be achieved by freeze-thawing or dehydration/rehydration or by reverse phase evaporation (Monnard PA et al, Biochem.
Biophys. Acta 1997 1329 39-50) or by other published methods of preparing liposomes.
Liposomes prepared as described above would be added to 1 x 106 isolated immature dendritic cells or to other antigen presenting cells at a concentration of 25 micrograms per ml. The maturation and function of the dendritic cells or other antigen presenting cells is assessed as previously described. The liposomes containing phosphatidylserine (with or without targeting molecules) is used to treat dendritic cells or other antigen presenting cells ex vivo or for systemic treatment.

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SUBSTITUTE SHEET (RULE 26) cd 36 in HDB-DI HDB-NOND1 http://www.atcc.org/cgi-bin/Sfgat...abase=local%2FHDB-NOND1&text=cd36 Your query was:
cd36 The selected databases contain 18 documents matching your query:
1: _1013396 RE 1.CE>platelet 1.SN>CD36 l.a.CC>differentia 2: 1003558 RE 1.CE>platelet 1.U>cell membrane 1.SN>CD36 3: 1018253 RE 1.SN>CD36 l.a.CC>differentiation l.b.CC>gly 4: 1020319 RE 1.G>Homo sapiens l.CN>human 1.SN>CD36 5: 1013397 RE l.CE>platelet 1.SN>CD36 l.a.CC>differentia 6: 1020540 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 7: 1017636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 8: 1022016 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 9: 1019865 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 10: 1024459 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 11: 1019119 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1.MW

12: 1009963 RE 1.G>Homo Sapiens 1.CN>human 1.CE>monocyte 13: 1016854 RE 1.G>Homo Sapiens 1.CN>human 1.CE>platelet 14: 1023242 RE 1.G>Homo Sapiens 1.CN>human l.U>cell membr 15: 22825 RE 1.G>Homo Sapiens 1.CN>human 1.CE>platelet 16: 1012440 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 17: 1012380 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 18: 1003358 RE 1.SN>ACTH 1.FS>N-terminal region l.a.CC>ho SUBSTITUTE SHEET (RULE 26) 013396 RE 1. CE>platelet 1.SN>CD36 l.a.CC>differentia 1013396 RE 1. CE>platelet 1.SN>CD36 l.a.CC>differentiation DI P>Medica DI 2382 Camino Vida Roble, Suite I
DI Carlsbad, CA 92009 USA

DE C>CLB/703 ;developer DE P>MON1118 ;distributor PD ;IgGl RE l.CE>platelet 1.SN>CD36 l.a.CC>differentiation RE 2.CE>monocyte 2.SN>CD36 2.a.CC>differentiation RE 3. CE>macrophage 3.SN>CD36 3.a.CC>differentiation RE 4. CE>platelet 4.SN>CD36 4.a.CC>differentiation AP ;frozen section LD USA BAL
EI DA>9303 CI ;catalog SN Synonym>CD36 1013396 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) _003558 RE 1. CE>platelet 1.U>cell membrane 1.SN>CD36 1003558 RE 1. CE>platelet 1.U>cell membrane 1.SN>CD36 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DE P>N42540M ;distributor PD ;IgGl RE 1. CE>platelet 1.U>cell membrane 1.SN>CD36 RE l.a.CC>differentiation l.b.CC>protein RE l.c.CC>blood coagulation factor AV ;purified LD USA BAL
EI DA>9002 CV>9007 CI ;catalog SN Synonym>CD36 1003558 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 018253 RE 1.SN>CD36 l.a.CC>differentiation l.b.CC>gl 1018253 RE 1.SN>CD36 l.a.CC>differentiation l.b.CC>glycoprotein DI P>BioGenex Laboratories DI 4600 Norris Canyon Road DI San Ramon, CA 94583 USA

DI 1-800-421-4149 (toll free USA) DE P>lE8 ;distributor DO G>Mus musculus CN>mouse RE 1.SN>CD36 l.a.CC>differentiation l.b.CC>glycoprotein AV ;purified LD USA JMJ
EI DA>9602 CI ;catalog SN Synonym>CD36 1018253 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 020319 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1020319 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 DI P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DE C>89 ;distributor DE P>MCA1214 ;distributor DO G>Mus musculus CN>mouse PD ;IgG2b RE 1.G>Homo sapiens l.CN>human 1.SN>CD36 RE l.a.CC>differentiation AP ;flow cytometry ;Western blot AV ;purified LD USA CLB
EI DA>9702 CI ;catalog SN Synonym>CD36 1020319 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 013397 RE 1. CE>platelet 1.SN>CD36 l.a.CC>differentia 1013397 RE 1. CE>platelet 1.SN>CD36 l.a.CC>differentiation DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I
DI Carlsbad, CA 92009 USA

DE C>VM58 ;developer DE P>MON1143 ;distributor DE P>VM58 ;distributor DE 2.P>MON1143 ;distributor PD ;IgGl RE 1. CE>platelet 1.SN>CD36 l.a.CC>differentiation RE 2. CE>monocyte 2.SN>CD36 2.a.CC>differentiation RE 3. CE>macrophage 3.SN>CD36 3.a.CC>differentiation RE 4. CE>platelet 4.SN>CD36 4.a.CC>differentiation AP ;frozen section LD USA BAL
EI DA>9303 CI ;catalog SN Synonym>CD36 1013397 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) _020540 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1020540 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 DI P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DE P>89 ;developer DE P>MCA1214 ;distributor DO G>Mus musculus CN>mouse PD ;IgG2b RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation AP ;flow cytometry ;Western blot AV ;purified LD USA CLB
EI DA>9702 CI ;catalog SN Synonym>CD36 1020540 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 117636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1017636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 DI P>Novocastra Laboratories Ltd.
DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK

DE P>NCL-CD36 ;distributor DE P>SMO ;distributor DO G>Mus musculus CN>mouse AS ;immunohistochemical staining RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation AP ;frozen section AV ;ascites SD SMO
LD USA BAL
EI DA>9904 CI ;catalog SN Synonym>CD36 1017636 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 022016 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1022016 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 DI P>O.E.M. Concepts, Inc.
DI 1889 Route 9, Bldg. 25, Unit 96 DI Toms River, NJ 08755 USA

DE C>289-10930 ;distributor DE P>M2-L69 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Homo sapiens 1.CN>human l.SN>CD36 RE l.a.CC>differentiation AP ;cell surface marker AV ;purified LD USA EJK
EI DA>9712 CI ;catalog SN Synonym>CD36 1022016 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 019865 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1019865 RE l.G>Homo Sapiens 1.CN>human l.SN>CD36 DI P>Upstate Biotechnology, Inc.
DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA

DI 1-800-233-3991 (toll free USA) (sales) DE P>05-287 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgM
RE 1.G>Homo Sapiens 1.CN>human l.SN>CD36 RE l.a.CC>differentiation l.b.CC>protein AP ;immunocytochemistry AV ;ascites LD USA JMJ
EI DA>9611 CI ;catalog SN Synonym>CD36 1019865 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 016854 RE l.G>Homo sapiens 1.CN>human 1. CE>platelet 1009963 RE 1.G>Homo Sapiens l.CN>human l.CE>monocyte 1009963 RE 1.G>Homo Sapiens 1.CN>human 1. CE>monocyte 1.SN>CD36 DI P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 2.P>Immunotech S.A.
DI Departement commercial DI Luminy Case 915 DI 13288 Marseille Cedex 9, France DE C>Fa6-152 ;developer DE P>MCA 682 ;distributor DE 2.P>0765 ;distributor DE 2.P>0766 ;distributor DE 2.P>FA6.152 ;distributor DO G>Mus musculus CN>mouse PD )IgGl RE l.G>Homo sapiens 1.CN>human 1. CE>monocyte 1.SN>CD36 RE l.a.CC>differentiation RE 2.G>Homo sapiens 2.CN>human 2. CE>macrophage 2.SN>CD36 RE 2.a.CC>differentiation RE 3.G>Homo sapiens 3.CN>human 3. CE>platelet 3.SN>CD36 RE 3.a.CC>differentiation AV ;purified ;2.fluorescein conjugate ;2. purified LD USA BAL
EI DA>9103 CV>9104 CI ;catalog SN Synonym>CD36 1009963 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) _023242 RE 1.G>Homo sapiens 1.CN>human 1.U>cell membr 1023242 RE 1.G>Homo sapiens 1.CN>human 1.U>cell membrane 1.SN>CD36 SO Exp Cell Res 1992;198:85-92 SO J Exp Med 1990;171:1883-92 DI P>Lab Vision-NeoMarkers DI 47770 Westinghouse Drive DI Fremont, CA 94539 USA

DE C>lA7 ;distributor DE P>MS-466-P ;distributor IM G>Homo sapiens CN>human CE>platelet SN>CD36 a.CC>differentiation IM b.CC>glycoprotein c.CC>receptor DO G>Mus musculus CN>mouse PD ;IgG2b ;kappa RE 1.G>Homo sapiens l.CN>human 1.U>cell membrane 1.SN>CD36 RE 1. MW>88 kD l.a.CC>differentiation l.b.CC>glycoprotein RE l.c.CC>receptor AP ;flow cytometry ;immunofluorescence ;immunoprecipitation AP ;Western blot ;immunohistology ;gold labelling AB platelet GPIIIb, platelet glycoprotein IIIb, and OKMS-antigen.

LD USA MCM
EI DA>9806 CI ;catalog SN Synonym>CD36 1023242 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) ?825 RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 22825 RE l.G>Homo Sapiens 1.CN>human 1. CE>platelet AU Kemshead J
AD Imperial Cancer Research Technology;
AD Sardinia House;
AD Sardinia Street;
AD London WC2A 3NL;
AD UK;
AD Tel Ol 242 1136;
AD TELEX 265107.TCRFG;

SO Br J Haematol 1984;57:621 DE P>M148 ;developer IM G>Homo Sapiens 1.CN>human PA>medulloblastoma a.CC>neoplasm PD ;IgGl RE l.G>Homo Sapiens 1.CN>human 1. CE>platelet RE 1.U>cell surface 1.SN>CD36 1.MW>110-130 kD
RE l.a.CC>differentiation RE 2.G>Homo Sapiens 2.CN>human 2. PA>medulloblastoma RE 2.a.CC>neoplasm RE 3.G>Homo sapiens 3.CN>human 3. PA>neuroblastoma RE 3.a.CC>neoplasm RE 4.G>Homo Sapiens 4.CN>human 4. PA>rhabdomyosarcoma RE 4.a.CC>neoplasm AP ;immunofluorescence ;immunoprecipitation AB in vivo imaging and therapy LD EUR BD FI>EUR0003951 EUR901.TXT
EI DA>8901 CV>8904 CI ;catalog SN Synonym>CD36 22825 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1012380 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1012380 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DI 2.P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 96229-0176 DI 3.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA

DI 4.P>Sigma Chemical Company DI P.O. Box 14508 DI St. Louis, MO 63178 9916 USA
DI 1-800-325-3010 (toll free USA) DE C>SMO ;developer DE P>P54168M ;distributor DE P>SMO ;distributor DE 2.P>MCA-722F ;discontinued designation DE 2.P>MCA722 ;distributor DE 2.P>SMO :distributor DE 3.P>LBL 268 ;distributor DE 3.P>SMO ;distributor DE 4.P>C 4679 ;distributor DE 9.P>F5898 ;distributor DE 4.P>P9312 ;distributor DE 4.P>R6395 ;distributor DE 4.P>SMO :distributor DO G>Mus musculus CN>mouse PD ;IgM
RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation AV ;purified ;2.purified ;4.fluorescein conjugate AV ;4.phycoerythrin conjugate SD SMO
SD SMO
LD USA BAL
EI DA>9803 Cv>9111 CI ;catalog SN Synonym>CD36 1012380 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1003558 RE 1. CE>platelet 1.U>cell membrane 1.SN>CD36 1003558 RE 1. CE>platelet 1.U>cell membrane 1.SN>CD36 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DE P>N42540M ;distributor PD ; IgGl RE 1. CE>platelet 1.U>cell membrane l.SN>CD36 RE l.a.CC>differentiation l.b.CC>protein RE l.c.CC>blood coagulation factor AV ;purified LD USA BAL
EI DA>9002 CV>9007 CI ;catalog SN Synonym>CD36 1003558 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1024459 RE.1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1024459 RE l.G>Homo Sapiens 1.CN>human 1.SN>CD36 DI P>Biogenesis Ltd.
DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK

DE C>SM-phi IgM ;distributor DE P>2125-3607 ;distributor DO G>Mus musculus CN>mouse PD ; Ig RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation AP ;immunofluorescence AV ;fluorescein conjugate AB CD36 is also known as platelet GPIV, GPIV, platelet GPIIIb, GPIIIb, platelet AB glycoprotein IV, and FAT (rat).

SD SMPHIIGM
LD USA MCM
EI DA>9811 CI ;catalog SN,Synonym>CD36 1024459 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1016854 RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 1016854 RE 1.G>Homo sapiens 1.CN>human 1. CE>platelet DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA

DI 1-800-848-6227 (toll free USA) DE P>CB38 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ;IgM ;kappa RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet RE 1.U>cell membrane 1.SN>CD36 1.MW>88 kD
RE l.a.CC>differentiation l.b.CC>glycoprotein AP ;flow cytometry ;immunoprecipitation AV ;fluorescein conjugate ;purified LD USA JMJ
EI DA>9504 CI ;catalog SN Synonym>CD36 1016854 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1012440 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1012440 RE 1.G>Homo Sapiens l.CN>human 1.SN>CD36 DI P>BioSource International DI 820 Flynn Roa DI Camarillo, CA 93012 USA
DI 1-800-242-0607(toll free USA) DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK

DI 3.P>Roche Molecular Biochemicals DI formerly Boehringer Mannheim GmbH
DI Sandhofer Strasse 116 DI D-68305 Mannheim Germany DE C>SMO ;developer DE P>AHS3601 ;distributor DE P>AHS3608 ;distributor DE P>CS-CD36-FI ;discontinued designation DE P>CS-CD36-UN ;discontinued designation DE P>SMO ;distributor DE 2.P>CBL 168 ;distributor DE 2.P>SMO ;distributor DE 3.P>1441 230 ;discontinued designation DE 3.P>1441 264 ;distributor DE 3.P>SMO ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation AV ;fluorescein conjugate ;3. purified SD SMO
LD USA BAL
EI DA>9709 CV>9111 CI ;catalog SN Synonym>CD36 1012440 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) 1003358 RE 1.SN>ACTH l.FS>N-terminal region l.a.CC>ho 1003358 RE 1.SN>ACTH 1. FS>N-terminal region l.a.CC>hormone DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK

DE C>58 ;developer DE P>E54008M ;distributor RE 1.SN>ACTH 1. FS>N-terminal region l.a.CC>hormone AV ;purified AB CD36 is also known as GPIIIb, GPIV

LD USA BAL
EI DA>9002 CV>9007 CI ;catalog SN Synonym>ACTH
SUBSTITUTE SHEET (RULE 26) 1019119 RE 1.G>Homo sapiens l.CN>human 1.SN>CD36 1. MW
1019119 RE 1.G>Homo Sapiens 1.CN>human l.SN>CD36 1.MW>88 kD

SO J Cell Biol 1994;269:6011 SO J Cell Biol 1993;268:16179 DI P>Transduction Laboratories DI 133 Venture Ct., Suite 5 DI Lexington, Ky 40511-9923 DE P>73 ;distributor DE P>C23620 ;distributor IM G>Homo Sapiens CN>human SN>CD36 FS>amino acids 70-242 IM a.CC>protein DO G>Mus musculus CN>mouse PD ;IgG2a RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD36 1.MW>88 kD
RE l.a.CC>protein RE 2.G>Rattus norvegicus 2.CN>Norway rat 2.SN>CD36 RE 2. MW>88 kD 2.a.CC>protein RE 3.G>Gallus gallus 3.CN>chicken 3.SN>CD36 3.MW>88 kD
RE 3.a.CC>protein AP ;Western blot ;immunofluorescence AV ;purified LD USA JMJ
EI DA>9901 CI ;catalog SN Synonym>CD36 1019119 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) DI is distributor SD is standard designation SUBSTITUTE SHEET (RULE 26) 1022961 RE l.SN>CD51 l.a.CC>differentiation 1022961 RE l.SN>CD51 l.a.CC>differentiation DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DE C>NGX-IV/110 ;distributor DE P>MON1027 ;distributor RE 1.SN>CD51 l.a.CC>differentiation LD USA MCM
EI DA>9805 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1022017 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51/61 c 1022017 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51/61 c DI P>O.E.M. Concepts,.Inc DI 1889 Route 9, Bldg. 25, Unit 96 DI Toms River, NJ 08755 USA

DE C>289-12336 ;distributor DE P>M2-L69 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51/61 complex RE l.a.CC>differentiation AP ;cell surface marker AV ;purified AB Reactant#l: CD51/61 complex is also known as integrin alpha V
beta 3.

LD USA EJK
EI DA>9712 CI ;catalog SN Synonym>CD51/61 complex 1022018 ****HB/HYBRID
SUBSTITUTE SHEET (RULE 26) 1013413 RE 1. CE>platelet 1.SN>CD51 l.a.CC>differentiation 1013413 RE 1. CE>platelet 1.SN>CD51 l.a.CC>differentiation DI P>Medica DI 2382 Camino Vida Roble, Suite I
DI Carlsbad, CA 92009 USA

DE C>706 ;developer DE P>MON1130 ;distributor PD ;IgGl RE 1. CE>platelet 1.SN>CD51 l.a.CC>differentiation LD USA BAL
EI DA>9303 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1024461 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1024461 RE 1.G>Homo sapiens 1.CN>human l.SN>CD51 DI P>Biogenesis Ltd.
DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK .

DE C>13C2 ;distributor DE P>2125-5108 ;distributor DE P>2125-5114 ;distributor DE P>2125-5119 ;distributor DO G>Mus musculus CN>mouse PD ; Ig RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation AP ;immunofluorescence AV ;R-phycoerythrin conjugate ;fluorescein conjugate AB CD51 is also known as integrin alpha V subunit and vitronectin receptor AB alpha subunit.

LD USA MCM
EI DA>9811 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1017037 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 1017037 RE l.G>Homo Sapiens 1.CN>human 1.SN>CD51 DI P>Zymed Laboratories Inc.
DI 458 Carlton Court DI South San Francisco, CA 94080 USA
DI 1-800-874-4494 (toll free USA) DE P>07-5103 ;distributor DE P>NK1-M9 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ; IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation l.b.CC>protein AP ;flow cytometry ;immunofluorescence AV ;purified LD USA JMJ
EI DA>9708 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1009962 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 1009962 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DI 2.P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DE C>AMF7 ;developer DE P>AMF7 ;distributor DE P>P42770M ;distributor DE 2.P>MCA 683 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation AV ;purified ;2.purified LD USA BAL
EI DA>9103 CV>9104 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1021411 RE 1.G>Homo sapiens 1.CN>human 1. CE>platelet 1021411 RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 1.SN>CD51 DI P>Immunotech S.A.
DI Departement commercial DI Luminy Case 915 DI 13288 Marseille Cedex 9, France DE C>69-6-5 ;distributor DE P>1603 ;distributor DO G>Mus musculus CN>mouse S>BALB/c 0>spleen PD ;IgG2a RE 1.G>Homo sapiens 1.CN>human 1. CE>platelet l.SN>CD51 RE l.a.CC>protein AV ;purified LD USA JMJ
EI DA>9707 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1015384 RE 1.G>Mus musculus 1.CN>mouse 1.SN>CD51 1015384 RE 1.G>Mus musculus 1.CN>mouse l.SN>CD51 DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA

DI 1-800-848-6227 (toll free USA) DE C>H9.2B8 ;developer DE P>01520D ;distributor DE P>01521D ;distributor DE P>01522D ;distributor DE P>01525B ;distributor DO G>Cricetulus sp. CN>hamster IP G>Mus musculus CN>mouse PD ;IgG
RE l.G>Mus musculus 1.CN>mouse 1.SN>CD51 RE l.a.CC>differentiation AP ;flow cytometry ;immunofluorescence AV ;biotin conjugate ;fluorescein conjugate AV ;phycoerythrin conjugate ;purified LD USA BAL
EI DA>9408 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1023962 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 1023962 RE 1.G>Homo sapiens 1.CN>human l.SN>CD51 SO Cell 1992;69:11-25 DI P>Ancell Corporation DI 243 Third Street North DI P.O. Box 87 DI Bayport, MN 55003 USA
DI 1-800-374-9523 (toll free USA) DE C>P2W7 ;distributor DE P>202-020 ;distributor DE P>202-030 ;distributor DE P>202-040 ;distributor DE P>202-050 ;distributor IM G>Homo Sapiens CN>human O>eye PA>melanoma CD>V+B2 a.CC>neoplasm DO G>Mus musculus CN>mouse PD ;IgGl ;kappa RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation AP ;immunoprecipitation ;flow cytometry ;frozen section AV ;R-phycoerythrin conjugate ;biotin conjugate AV ;fluorescein conjugate ;purified LD USA MCM
EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) _ 77 1023559 RE 1.G>Mus musculus 1.CN>mouse 1.SN>integrin 1023559 RE 1.G>Mus musculus 1.CN>mouse 1.SN>integrin alpha V
SO Biochemistry 1990;29:10191 SO Exp Cell Res 1993;205:25 DI P>Upstate Biotechnology, Inc.
DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA

DI 1-800-233-3991 (toll free USA) (sales) DE P>05-437 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Mus musculus l.CN>mouse 1.SN>integrin alpha V
RE 1. MW>160 kD l.a.CC>differentiation l.b.CC>receptor AP ;Western blot ;immunoprecipitation ;immunohistochemistry AV ;ascites AB Reactant is also known as vitronectin receptor alpha subunit and CD51.

LD USA MCM
EI DA>9807 CI ;catalog SUBSTITUTE SHEET (RULE 26) _ 78 _ 1023927 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin 1023927 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V

SO J Biol Chem 1994;269:6940 DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE C>P3G8 ;distributor DE P>MAB1953 ;distributor IM G>Homo sapiens CN>human 0>lung PA>carcinoma a.CC>neoplasm DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V
RE l.a.CC>differentiation l.b.CC>receptor AP ;immunocytology ;immunohistochemistry ;immunoprecipitation AP ;flow cytometry ;ELISA ;FACS
AV ;purified AB Reactant is also known as CD51 and vitronectin receptor alpha subunit.
AB Product reacts with all alpha V-containing integrin receptors.
AB Product will react with some lymphoid cell lines (B cells), many carcinoma and AB melanoma cell lines and osteosarcomas.

LD USA MCM
EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) _ 79 _ 1015432 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1. FS
1015432 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1. FS>alpha subunit DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA

DI 2.P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 3.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK

DI 4.P>Endogen Inc.
DI 30 Commerce Way DI Woburn, MA 01801-1059 USA

DI 5.P>Genosys Biotechnologies, Inc.
DI 1442 Lake Front Circle, Suite 185 DI The Woodlands, TX 77380-3600 USA

DI 1-800-234-5362 (toll free USA) DI 6.P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 7.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA

DI 8.P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA

DI 1-800-848-6227 (toll free USA) -DI 9.P>T Cell Diagnostics, Inc.
DI 6 Gill Street DI Woburn, MA 01801-1721 USA

DE C>23C6 ;developer DE P>23C6 ;distributor DE P>P54490M ;distributor DE 2.P>23C6 ;distributor DE 2.P>MON1167 ;distributor DE 3.P>23C6 ;distributor DE 3.P>CBL490 ;distributor DE 4.P>23C6 ;distributor DE 4.P>MA-5100 ;distributor DE 5.P>23C6 ;distributor DE 5.P>AM-19-760 ;distributor DE 6.P>23C6 ;distributor DE 6.P>MCA-757 ;discontinued designation SUBSTITUTE SHEET (RULE 26) DE 6.P>MCA757G ;distributor DE 7.P>23C6 ;distributor DE 7.P>LBL 590 ;distributor DE 8.P>23C6 ;distributor DE 8.P>31561A ;distributor DE 8.P>31564X ;distributor DE 9.P>23C6 ;distributor DE 9.P>IA1S04 ;distributor DO G>Mus musculuS CN>mouse PD ; IgGl RE l.G>Homo sapiens 1.CN>human 1.SN>CD51 1. FS>alpha subunit RE 1. MW>125 kD l.a.CC>differentiation AV ;purified ;4.purified ;6. purified ;8.fluorescein conjugate AV ;8.purified ;9. supernatant LD USA BAL
EI DA>9706 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011348 RE 1.G>Homo Sapiens 1.CN>human 1.SN>vitronect 1011348 RE l.G>Homo Sapiens 1.CN>human 1.SN>vitronectin receptor DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE P>CLB-706 ;distributor DE P>MAB1980 ;distributor RE 1.G>Homo Sapiens 1.CN>human l.SN>vitronectin receptor RE l.a.CC>receptor AV ;purified AB beta subunit of vitronectin receptor referred to as CD51 also AB Reactant#1: vitronectin receptor beta subunit syn. for CD51 LD USA BAL
EI DA>9107 CV>9108 CI ;catalog SN Synonym>vitronectin receptor SUBSTITUTE SHEET (RULE 26) DI is distributor SD is standard designation SUBSTITUTE SHEET (RULE 26) Beta 5 1019741 RE 1.G>Homo Sapiens 1.CN>human l.SN>integrin beta 5 DI P>Upstate Biotechnology, Inc.
DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA

DI 1-800-233-3991 (toll free USA) (sales) DE C>B5-IVF2 ;distributor DE P>05-283 ;distributor DO G>Mus musculus CN>mouse S>BALB/c 0>spleen PD ;IgGl RE 1.G>Homo Sapiens l.CN>human 1.SN>integrin beta 5 RE l.a.CC>protein AP ;immunocytochemistry ;blocks cell adhesion AV ;ascites LD USA JMJ
EI DA>9611 CI ;catalog SUBSTITUTE SHEET (RULE 26) Beta 3 1011332 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE P>MAB1974 ;distributor PD ; IgGl RE-1.G>Homo Sapiens l.CN>human 1.SN>integrin beta 3 RE l.a.CC>receptor AV ;ascites LD USA BAL
EI DA>9107 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1014236 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin beta 3 AU Ylanne J
SO Blood 1989;72:1478-86 SO Blood 1990;76:570-7 DI P>Biohit OY
DI Verkkosaarenkatu 4 DI 00580 Helsinki, Finland DI 2.P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 3.P>ICN Biomedicals DI Biomedical Research Products DI 3300 Hyland Avenue DI Costa Mesa, CA 92626 DI 1-800-854-0530 (toll free USA) DI 4.P>Locus Genex Oy DI Verkkosaarenkatu 4 DI 00580 Helsinki, Finland DE C>BB10 ;developer DE P>M-9006000 ;distributor DE P>M-9006100 ;distributor DE 2.P>MCA-781 ;distributor DE 2.P>bbl0 ;distributor DE 3.P>69-323-1 ;distributor DE 3.P>69-323-2 ;distributor DE 3.P>BB10 ;distributor DE 4.P>BB10 ;distributor DE 4.P>M-9006000 ;distributor DE 4.P>M-9006100 ;distributor IM G>Homo Sapiens CN>human SN>CD41 ;purified a.CC>differentiation RM ;in vivo DO G>Mus musculus CN>mouse S>BALB/c 0>spleen IP G>Mus musculus CN>mouse PA>myeloma PD ;IgGl RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin beta 3 RE l.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2.SN>platelet GPIIIa RE 2.a.CC>blood coagulation factor AP ;immunoassay ;not paraffin section ;immunohistochemical staining AP ;immunoblotting AV ;purified ;2.purified ;3. purified ;4. purified LD USA BAL
SUBSTITUTE SHEET (RULE 26) EI DA>9711 SN Synonym>CD41 1014236 SN Synonym>CD41 SUBSTITUTE SHEET (RULE 26) _ 87 1014281 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 DI P>Bio-Science Products AG
DI Gerliswilstrasse 93 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DE P>0121022 ;distributor RE 1.G>Homo sapiens 1.CN>human l.SN>integrin beta 3 RE l.a.CC>receptor RE 2.G>Homo Sapiens 2.CN>human 2.SN>CD41 RE 2.a.CC>differentiation AP ;immunoassay ;immunoblotting ;not paraffin section AP ;immunohistochemical staining LD USA BAL
EI DA>9305 CI ;catalog SN Synonym>CD41 1014281 SN Synonym>CD41 SUBSTITUTE SHEET (RULE 26) _ 88 _ 1019109 RE 1.G>Homo Sapiens l.CN>human 1.SN>integrin beta 3 SO J Cell Biol 1993;122:223 SO J Cell Biol 1993;121:689 DI P>Transduction Laboratories DI 133 Venture Ct., Suite 5 DI Lexington, Ky 40511-9923 DE P>26 ;distributor DE P>I19620 ;distributor IM G>Mus musculus CN>mouse SN>integrin beta 3 FS>amino acids 16-223 IM a.CC>protein DO G>Mus musculus CN>mouse PD ;IgM
RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin beta 3 RE 1. MW>90 kD l.a.CC>protein RE 2.G>Canis familiaris 2.CN>dog 2.SN>integrin beta 3 RE 2. MW>90 kD 2.a.CC>protein RE 3.G>Rattus norvegicus 3.CN>Norway rat 3.SN>integrin beta 3 RE 3. MW>90 kD 3.a.CC>protein RE 4.G>Mus musculus 4.CN>mouse 4.SN>integrin beta 3 RE 4. MW>90 kD 4.a.CC>protein RE 5.G>Gallus gallus S.CN>chicken S.SN>integrin beta 3 RE 5. MW>90 kD S.a.CC>protein AP ;Western blot AV ;purified LD USA JMJ
EI DA>9705 CI ;catalog SUBSTITUTE SHEET (RULE 26) _ 89 _ 1023930 RE 1.G>Homo Sapiens 1.CN>human l.SN>integrin beta 3 SO Cell 1986;45:269-80 DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE C>25E11 ;distributor DE P>MAB1957 ;distributor IM T>blood CE>mononuclear cell CS>activated DO G>Mus musculus CN>mouse PD ;IgG2a RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin beta 3 RE l.a.CC>differentiation l.b.CC>receptor RE 2.SN>CD41/CD61 complex 2.a.CC>protein RE 3.G>Homo Sapiens 3.CN>human 3. CE>monoblast 3. PA>leukemia RE 3. CD>U937 3.SN>CD41/CD61 complex 3.a.CC>protein AP ;Western blot ;immunocytology ;immunoprecipitation AP ;flow cytometry AV ;purified a.PM>protein A chromatography AB Integrin beta 3 is also known as CD61, GPIIIa, and vitronectin AB receptor beta chain.

LD USA MCM
EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1024059 RE 1.G>Homo sapiens 1.CN>human 1. CE>platelet l.SN>CD61 DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA

DI 1-800-848-6227 (toll free USA) DE C>VI-PL2 ;distributor DE P>33821 ;distributor DE P>33824 ;distributor DE P>33825 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl ;kappa RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 1.SN>CD61 RE 1. MW>105 kD l.a.CC>differentiation l.b.CC>glycoprotein RE 2.G>Homo Sapiens 2.CN>human 2. CE>megakaryocyte 2.SN>CD61 RE 2. MW>105 kD 2.a.CC>differentiation 2.b.CC>glycoprotein RE 3.G>Homo sapiens 3.CN>human 3. CE>osteoclast 3.SN>CD61 RE 3. MW>105 kD 3.a.CC>differentiation 3.b.CC>glycoprotein RE 4.G>Homo Sapiens 4.CN>human 4.T>endothelium 4.SN>CD61 RE 4.MW>105 kD 4.a.CC>differentiation 4.b.CC>glycoprotein XR 1.G>Canis sp. 1.CN>dog 1.SN>CD61 l.a.CC>differentiation XR l.b.CC>glycoprotein XR 2.G>Felis Sp. 2.CN>cat 2.SN>CD61 2.a.CC>differentiation XR 2.b.CC>glycoprotein NR 1.G>Sus Sp. 1.CN>swine 1.SN>CD61 l.a.CC>differentiation NR l.b.CC>glycoprotein AP ;acetone fixed ;frozen section AV ;R-phycoerythrin conjugate ;biotin conjugate AV ;fluorescein conjugate ;purified AB CD61 is also known as integrin beta 3 subunit.

LD USA MCM
EI DA>9809 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011347 RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE P>MAB1984 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor RE l.a.CC>receptor AB Reactant#1 vitronectin receptor alpha subunit syn. for CD61 LD USA BAL
EI DA>9107 CV>9108 CI ;catalog SN Synonym>vitronectin receptor SUBSTITUTE SHEET (RULE 26) 1012445 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 DI P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK

DE C>Thromb/1 ;developer DE P>CBL 458 ;discontinued designation RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation LD USA BAL
EI DA>9110 CV>9111 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1013416 RE 1. CE>platelet 1.SN>CD61 l.a.CC>differentiation DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I
DI Carlsbad, CA 92009 USA

DI 3.P>Sigma Chemical Company DI P.O. Box 14508 DI St. Louis, MO 63178 9916 USA
DI 1-800-325-3010 (toll free USA) DE C>BL-E6 ;developer DE P>BL-E6 ;distributor DE P>MHCD6101 ;distributor DE P>MHCD6101-4 ;distributor DE P>MHCD6115 ;distributor DE P>MHCD6115-4 ;distributor DE P>MON1051 ;distributor DE 2.P>MON1051 ;distributor DE 3.P>BL-E6 ;distributor DE 3.P>C4321 ;distributor DE 3.P>F7902 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1. CE>platelet 1.SN>CD61 l.a.CC>differentiation RE 2. CE>megakaryocyte 2.SN>CD61 2.a.CC>differentiation AP ;frozen section AV ;biotin conjugate ;fluorescein conjugate AV ;3.fluorescein conjugate LD USA BAL
EI DA>9803 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1013417 RE 1. CE>platelet 1.SN>CD61 l.a.CC>differentiation DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, 'CA 94010 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I
DI Carlsbad, CA 92009 USA

DE C>CRC54 ;developer DE P>CRC54 ;distributor DE P>MON1147 ;distributor DE 2.P>MON1147 ;distributor PD ; IgGl RE 1. CE>platelet 1.SN>CD61 l.a.CC>differentiation RE 2. CE>megakaryocyte 2.SN>CD61 2.a.CC>differentiation AP ;frozen section LD USA BAL
EI DA>9303 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1014017 RE 1.G>Homo Sapiens 1.CN>human 1.SN>CD61 DI P>Diagast Laboratories DI 59, rue de Trevise-B.P. 2034 , DI 59014 Lille Cedex, France DI DIAGAST (042) 160716F
DE P>16101V ;distributor DE P>16103A ;distributor DE P>16105E ;distributor PD ;IgG
RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation AV ;fluorescein conjugate ;phycoerythrin conjugate ;purified LD USA BAL
EI DA>9304 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1014618 RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 1.SN>CD61 DI P>BioSource International DI 820 Flynn Roa DI Camarillo, CA 93012 USA
DI 1-800-242-0607(toll free USA) DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southamptoh, Hampshire S01 7NS UK

DI 3.P>Endogen Inc.
DI 30 Commerce Way DI Woburn, MA 01801-1059 USA

DI 4.P>Genosys Biotechnologies, Inc.
DI 1442 Lake Front Circle, Suite 185 DI The Woodlands, TX 77380-3600 USA

DI 1-800-234-5362 (toll free USA) DI 5.P>Harlan Bioproducts for Science, Inc.
DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 6.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA

DI 7.P>Novocastra Laboratories Ltd.
DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK

DI 8.P>Southern Biotechnology Associates, Inc.
DI P.O. Box 26221 DI Birmingham, AL 35260 USA
DI 1-800-722-2255 (toll free USA) DI 9.P>T Cell Diagnostics, Inc.
DI 6 Gill Street DI Woburn, MA 01801-1721 USA

DE C>PM 6/13 ;developer DE P>AHS6101 ;distributor SUBSTITUTE SHEET (RULE 26) DE P>AHS6107 ;distributor DE P>AHS6108 ;distributor DE P>CS-CD61-CF ;discontinued designation DE P>CS-CD61-FI ;discontinued designation DE P>CS-CD61-PE ;discontinued designation DE 2.P>CBL479 ;distributor DE 2.P>PM6/13 ;distributor DE 3.P>MA-6100 ;distributor DE 3.P>PM6/13 ;distributor DE 4.P>AM-19-705 ;distributor DE 4.P>PM6/13 ;distributor DE 5.P>MCA-728 ;distributor ' DE 5.P>MCA-728F ;distributor DE 5.P>MCA-728PE ;distributor DE 5.P>PM6/13 ;distributor DE 6.P>LBL 579 ;distributor DE 6.P>PM6/13 ;distributor DE 7.P>NCL-CD61 ;distributor DE 7.P>PM6/13 ;distributor DE 8.P>9470-O1 ;distributor DE 8.P>9470-02 ;distributor DE 8.P>9470-08 ;distributor DE 8.P>PM6/13 ;distributor DE 9.P>IA1S09 ;distributor DE 9.P>PM6/13 ;distributor DO G>Mus musculus CN>mouse PD :IgGl RE 1.G>Homo Sapiens 1.CN>human l.CE>platelet l.SN>CD61 RE 1. MW>90 kD l.a.CC>differentiation RE 2.G>Homo Sapiens 2.CN>human 2.T>plasma 2.SN>CD61 RE 2. MW>90 kD 2.a.CC>differentiation RE 3.G>Homo sapiens 3.CN>human 3.T>plasma RE 3.PA>unspecified neoplasm 3.SN>CD61 3.MW>90 kD
RE 3.a.CC>neoplasm 3.b.CC>differentiation AV ;fluorescein conjugate ;phycoerythrin conjugate ;purified AV ;3.purified ;5.fluorescein conjugate AV ;5.phycoerythrin conjugate ;5. purified ;8. biotin conjugate AV ;8.fluorescein conjugate ;8. purified ;9. purified SUBSTITUTE SHEET (RULE 26) LD USA BAL
EI DA>9904 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1017042 RE 1.G>Homo Sapiens l.CN>human 1. CE>platelet 1.SN>CD61 DI P>Becton Dickinson Immunocytometry Systems DI 2350 Qume Drive DI San Jose, CA 95131-1807 DI 1-800-223-8226 (toll free USA) DE P>348090 ;distributor DE P>348093 ;distributor DE P>559936 ;distributor DE P>RUU-PL7F12 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ;IgGl ;kappa RE 1.G>Homo Sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE l.a.CC>differentiation l.b.CC>protein AP ;immunofluorescence ;immunoprecipitation AV ;fluorescein conjugate ;purified LD USA JMJ
EI DA>9505 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1017635 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 DI P>Novocastra Laboratories Ltd.
DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK

DE P>NCL-CD61 ;distributor DE P>PM6/13 ;distributor DO G>Mus musculus CN>mouse AS ;immunohistochemical staining RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation AP ;frozen section AV ;ascites LD USA BAL
EI DA>9904 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1019745 RE 1.G>Homo sapiens l.CN>human 1.SN>CD61 DI P>Upstate Biotechnology, Inc.
DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA

DI 1-800-233-3991 (toll free USA) (sales) DE P>05-275 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ; IgGl RE l.G>Homo sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation l.b.CC>protein AP ;immunocytochemistry AV ;ascites LD USA JMJ
EI DA>9611 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1020416 RE 1.G>Homo Sapiens l.CN>human 1. CE>platelet 1.SN>CD61 DI P>PanVera Corporation DI 545 Science Drive DI Madison, WI 53711 USA

DE C>PL8-5 ;distributor DE P>TAK M068 ;distributor DO G>Mus musculus CN>mouse S>BALB/c 0>spleen PD ; IgG
RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet 1.SN>CD61 RE l.a.CC>differentiation l.b.CC>protein AV ;purified LD USA JMJ
EI DA>9702 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1020417 RE 1.G>Homo Sapiens 1.CN>human 1. CE>platelet l.SN>CD61 DI P>PanVera Corporation DI 545 Science Drive DI Madison, WI 53711 USA

DE C>PL11-7 ;distributor DE P>TAK M069 ;distributor DO G>Mus musculus CN>mouse S>BALB/c 0>spleen PD ;IgG
RE l.G>Homo Sapiens 1.CN>human 1. CE>platelet 1.SN>CD61 RE l.a.CC>differentiation l.b.CC>protein AV ;purified LD USA JMJ
EI DA>9702 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1021005 RE 1.G>Homo Sapiens 1.CN>human l.CE>platelet 1.SN>CD61 DI P>Cortex Biochem, Inc.
DI 1933 Davis Street, Suite 321 DI San Leandro, CA 94577 USA
DI 1-800-888-7713 (toll free USA) DI 1-510-568-3911(technical) DE P>CR1153 ;distributor RE 1.G>Homo Sapiens l.CN>human 1. CE>platelet 1.SN>CD61 RE l.a.CC>differentiation RE 2.G>Homo Sapiens 2.CN>human 2. CE>megakaryocyte 2.SN>CD61 RE 2.a.CC>differentiation LD USA CLB
EI DA>9704 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011328 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V

DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DI 2.P>Life Technologies, Inc.
DI 8400 Helgerman Ct.
DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA

DI 1-800-828-6686 (Toll free USA) DE P>MAB1958 ;distributor DE P>VNR147 ;distributor DE 2.P>12084-018 ;distributor DE 2.P>VNR147 ;distributor PD ; IgGl RE 1.G>Homo Sapiens l.CN>human 1.SN>integrin alpha V
RE l.a.CC>receptor AV ;ascites ;2.ascites LD USA BAL
EI DA>9706 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011329 RE 1.G>Homo Sapiens l.CN>human 1.SN>integrin alpha V

DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DI 2.P>Life Technologies, Inc.
DI 8400 Helgerman Ct.
DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA

DI 1-800-828-6686 (Toll free USA) DE P>MAB1960 ;distributor DE P>VNR139 ;distributor DE 2.P>12085-015 ;distributor DE 2.P>VNR139 ;distributor PD ;IgGl RE 1.G>Homo Sapiens 1.CN>human l.SN>integrin alpha V
RE l.a.CC>receptor AV ;ascites ;2.ascites LD USA BAL
EI DA>9706 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011842 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V

DI P>Biogenesis Ltd.
DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK

DE C>lU4/1 ;developer DE P>5355-2505 ;distributor RE l.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V
RE l.a.CC>protein AP ;immunoblotting AV ;ascites LD USA BAL
EI DA>9109 CV>9110 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1011843 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V

DI P>Biogenesis Ltd.
DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK

DE C>lU3/0 ;developer DE P>5355-2515 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V
RE l.a.CC>protein AP ;immunofluorescence AV ;ascites LD USA BAL
EI DA>9109 CV>9110 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1012705 RE l.G>Homo Sapiens 1.CN>human l.SN>integrin alpha V

DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DE P>LM142 ;distributor DE P>MAB1978 ;distributor PD ;IgG
RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V
RE l.a.CC>protein AV ;ascites LD USA BAL
EI DA>9112 CV>9201 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1013223 RE 1.SN>integrin alpha V l.a.CC>protein DI P>American Qualex International, Inc.
DI 920-A Calle Negocio St.
DI San Clemente, CA 92673 DI 1-800-772-1776 (toll free USA) DE P>M2580 ;distributor RE 1.SN>integrin alpha V l.a.CC>protein LD USA BAL
EI DA>9204 CV>9204 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1013510 RE 1.G>Homo Sapiens 1.CN>human 1. CE>endothelial cell AU Freed E
SO EMBO J 1989;8:2955 DI P>Calbiochem Novabiochem International DI P.O. Box 12087 DI La Jolla, CA 92039-2087 DI 1-800-854-3417(toll free USA) DE P>407281 ;distributor PD ;IgGl RE 1.G>Homo sapiens l.CN>human 1. CE>endothelial cell RE 1.SN>integrin alpha V 1. FS>type 1 l.a.CC>protein RE 2.G>Homo Sapiens 2.CN>human 2.SN>vitronectin RE 2.a.CC>protein RE 3.G>Homo sapiens 3.CN>human 3.SN>fibrinogen RE 3.a.CC>protein RE 4.G>Homo sapiens 4.CN>human 4.SN>osteopontin RE 4.a.CC>protein RE 5.G>Homo Sapiens S.CN>human 5.SN>von Willebrand factor RE 5.a.CC>protein RE 6.G>Homo Sapiens 6.CN>human 6.0>bone 6.SN>sialoprotein I
RE 6.a.CC>protein AP ;ELISA ;immunofluorescence ;immunoprecipitation AV ;ascites ;lyophilized LD USA BAL
EI DA>9303 CI ;catalog SN Synonym>fibrinogen SN Synonym>von Willebrand factor SUBSTITUTE SHEET (RULE 26) 1013511 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V

DI P>Calbiochem Novabiochem International DI P.O. Box 12087 DI La Jolla, CA 92039-2087 DI 1-800-854-3417(toll free USA) DE P>407282 ;distributor PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V
RE 1. FS>type 2 l.a.CC>protein RE 2.G>Homo Sapiens 2.CN>human 2.SN>vitronectin RE 2.a.CC>protein RE 3.G>Homo Sapiens 3.CN>human 3.SN>fibrinogen RE 3.a.CC>protein RE 4.G>Homo sapiens 4.CN>human 4.SN>osteopontin RE 4.a.CC>protein RE 5.G>Homo Sapiens S.CN>human 5.SN>von Willebrand factor RE S.a.CC>protein RE 6.G>Homo sapiens 6.CN>human 6.0>bone 6.SN>sialoprotein I
RE 6.a.CC>protein AP ) ELISA ; immunoblotting AV ;ascites ;lyophilized LD USA BAL
EI DA>9303 CI ;catalog SN Synonym>fibrinogen SN Synonym>von Willebrand factor SUBSTITUTE SHEET (RULE 26) 1014225 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V beta 5 AU Wayner EA
SO J Cell Biol 1991;113:919 DI P>Becton Dickinson Immunocytometry Systems DI 2350 Qume Drive DI San Jose, CA 95131-1807 DI 1-800-223-8226 (toll free USA) DI 2.P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500(toll free USA) DI 3.P>Life Technologies, Inc.
DI 8400 Helgerman Ct.
DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA

DI 1-800-828-6686 (Toll free USA) DI 4.P>Telios Pharmaceuticals, Inc.
DI 4757 Nexus Centre Drive DI San Diego, CA 92121 USA

DE C>P1F6 ;developer DE P>550045 ;distributor DE P>P1F6 ;distributor DE 2.P>MAB1961 ;distributor DE 2.P>P1F6 ;distributor DE 3.P>12078-O10 ;distributor DE 3.P>P1F6 ;distributor DE 4.P>A035 ;distributor DO G>Mus musculus CN>mouse PD ; IgGl RE 1.G>Homo Sapiens l.CN>human 1.SN>integrin alpha V beta 5 RE l.a.CC>receptor AP ;immunofluorescence ;immunoprecipitation AV ;2.ascites ;3.ascites ;4.ascites LD USA BAL
EI DA>9706 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1014277 RE 1.G>Homo Sapiens 1.CN>human l.CE>endothelial cell DI P>Bio-Science Products AG
DI Gerliswilstrasse 43 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DE P>0121005 ;distributor RE 1.G>Homo Sapiens l.CN>human 1. CE>endothelial cell RE 1.SN>integrin alpha V l.a.CC>receptor RE 2.G>Homo Sapiens 2.CN>human 2. PA>carcinoma RE 2.SN>integrin alpha V 2.a.CC>neoplasm 2.b.CC>receptor NR 1.G>Homo sapiens 1.CN>human 1.SN>fibronectin receptor NR l.a.CC>receptor NR 2.G>Homo Sapiens 2.CN>human 2.SN>CD41 NR 2.a.CC>differentiation AP ;ELISA ;immunofluorescence ;not immunoblotting AP ;immunoprecipitation LD USA BAL
EI DA>9305 CI ;catalog SN Synonym>CD41 1014277 SN Synonym>CD41 SN Synonym>fibronectin receptor SUBSTITUTE SHEET (RULE 26) 1014278 RE 1.G>Homo Sapiens 1.CN>human 1. CE>endothelial cell DI P>Bio-Science Products AG
DI Gerliswilstrasse 43 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DE P>0121006 ;distributor AS ; immunoblot RE 1.G>Homo Sapiens 1.CN>human 1. CE>endothelial cell RE l.SN>integrin alpha V l.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2. PA>carcinoma RE 2.SN>integrin alpha V 2.a.CC>neoplasm 2.b.CC>receptor AP ; immunoassay LD USA BAL
EI DA>9305 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1023559 RE 1.G>Mus musculus 1.CN>mouse 1.SN>integrin alpha V
SO Biochemistry 1990;29:10191 SO Exp Cell Res 1993;205:25 DI P>Upstate Biotechnology, Inc.
DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA

DI 1-800-233-3991 (toll free USA) (sales) DE P>05-437 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Mus musculus 1.CN>mouse l.SN>integrin alpha V
RE 1. MW>160 kD l.a.CC>differentiation l.b.CC>receptor AP ;Western blot ;immunoprecipitation ;immunohistochemistry AV ;ascites AB Reactant is also known as vitronectin receptor alpha subunit and CD51.

LD USA MCM
EI DA>9807 CI ;catalog SUBSTITUTE SHEET (RULE 26) 1023927 RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V

SO J Biol Chem 1994;269:6940 DI P>Chemicon International, Inc.
DI 28835 Single Oak Dr.
DI Temecula, CA 92590 USA

DI 1-800-437-7500 (toll free USA) DE C>P3G8 ;distributor IM G>Homo sapiens CN>human O>lung PA>carcinoma a.CC>neoplasm DO G.Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo Sapiens 1.CN>human 1.SN>integrin alpha V
RE l.a.CC>differentiation l.b.CC>receptor AP ;immunocytology ;immunohistochemistry ;immunoprecipitation AP ;flow cytometry ;ELISA ;FACS
AV ;purified AB Reactant is also known as CD51 and vitronectin receptor alpha subunit.
AB Product reacts with all alpha V-containing integrin receptors.
AB Product will react with some lymphoid cell lines (B cells), many AB carcinoma and melanoma cell lines and osteosarcomas.

LD USA MCM
EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) ~

SEQUENCE LISTING
<110> ISIS INNOVATION LTD
<i20> INDUCTION OF IMMUNE TOLERANCE
<130> SCB/52945/001 <140> PCT/GB00/02546 <i4i> 2000-06-30 <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <21i> 444 <212> PRT
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Claims

CLAIMS:

1. A method of treating mammalian dendritic cells in vitro to induce immune tolerance therein which comprises exposing said cells to an agonist of the cell surface receptors CD36 and/or CD51 as expressed on mammalian dendritic cells;
wherein said agonist is selected from: an antibody with an affinity for an epitope of CD36, an antibody with an affinity for an epitope of CD51, the Plasmodium falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1, thrombospondin, apoptotic cells or a negatively charged phospholipid.

2. A method as claimed in claim 1 wherein said dendritic cells are human or mouse.

3. A method as claimed in claim 1 or 2 which comprises exposing said dendritic cells to two or mare of the agonists of claim 1.

4. A method as claimed in any one of claims 1 to 3 wherein the pf-EMP-1 active binding domain comprises the amino acid sequence shown in Figure 2.

5. A method as claimed in any one of claims 1 to 4 wherein said cells are exposed to an antigenic material selected from: an auto-antigen associated with a particular auto-immune disease, an allo-antigen, a xeno-antigen or a therapeutic substance which is likely to induce an unwanted therapeutic response.

6. A method as claimed in claim 5 wherein the cells so produced are subsequently matured by exposure to an immune stimulus.

7. A method as claimed in any of claims 1 to 6 wherein said dendritic cells are prepared from human peripheral blood and or derived from CD34+ stem cells or monocytes.

8. A dendritic cell preparation obtainable by the method of any of claims 1 to 7 for use as a medicament.

9. A dendritic cell preparation obtainable by the method of any of claims 1 to 7 for use in inducing peripheral immune tolerance in a human.

10. A method of identifying a molecule which is an agonist of cell surface receptor CD36 and/or CD51 as expressed by mammalian dendritic cells which method comprises:
a) exposing immature mammalian dendritic cells to the molecule to be tested, b) exposing said immature dendritic cells to an immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, wherein impaired maturation in response to the immune stimulus is an indication that said molecule under test is a potential CD36 and/or CD51 agonist;
wherein if said test molecule is found to be a potential agonist of CD36 and/or CD51 the method further comprises the step of exposing said molecule to a purified sample of CD36 and/or CD51 and detecting any direct binding between said molecule and CD36and/or CD51.

11. A method as claimed in claim 10 wherein said dendritic cells are human cells or mouse cells.

12. A method as claimed in claim 10 or 11 wherein maturation of said dendritic cells is determined by examining the antigen-presenting ability of said cells.

13. A method as claimed in any of claims 10 to 12 wherein maturation of said dendritic cells is determined by examining said cells for expression of at least one cell surface antigen whose level of expression is enhanced in response to an immune stimulus.

14. A method as claimed in claim 13 wherein maturation of said dendritic cells is determined by measuring the level of expression of one or more of the following panel of antigens:
HLA DR, CD54, CD40, CD83 and CD86.

15. A method as claimed in claim 14 wherein said cells are also examined for expression of CD80.

16. A method as claimed in any one of claims 13 to 15 wherein the level of expression of said antigens is detected using a labelled antibody.

17. A method as claimed in 10 or 11 wherein maturation of said dendritic cells is determined by measuring said cells ability to induce T-cell proliferation.

18. The method of claim 10 or 11 wherein maturation of said dendritic cells is determined by quantifying the level of cytokines secreted from said cells:

19. The method of claim 18 wherein the level of secretion of TNF.alpha., IL12P70 and,IL10 is measured.

20. A method as claimed in any one of claims 10 to 19 wherein said immune stimulus is lipopolysaccharide, TNF.alpha., CD40L or monocyte conditioned medium (MCM).

21. A method as claimed in any one of claims 10 to 20 wherein said purified CD36 or CD51 is immobilised to a solid surface.

22. A method as claimed in anyone of claims 10 to 21 wherein said molecule is labelled with a detectable label.

23. A method as claimed in any one of claims 10 to 22 which further comprises the step of exposing said molecule to a purified sample of a v .beta.3 or a v .beta.5 and detecting any direct binding between said molecule and said a v .beta.3 or a v .beta.5.

24. A method as claimed in any one of claims 10 to 23 which further comprises the step of exposing said molecule to a purified sample of thrombospondin and detecting any direct binding between said molecule and thrombospondin.

25. A method as claimed in claim 23 or claim 24 wherein said molecule is labelled with a detectable label.

26. A method as claimed in any of claims 23 to 25 wherein said a v .beta.3, a v .beta.5 or thrombospondin is immobilised to a solid surface.

27. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD36 as expressed on mammalian dendritic cells and a pharmacologically acceptable carrier or diluent.

28. A composition as claimed in claim 27 which is suitable for inducing peripheral immune tolerance in a human wherein said CD36 agonist is selected from:
an antibody with an affinity for an epitope of CD36, the Plasmodium falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1 for CD36 and/or thrombospondin, thrombospondin, apoptotic cells or a negatively-charged phospholipid.

29. A composition as claimed in claim 28 wherein the pf-EMP-1 binding domain comprises the amino acid sequence as shown in Figure 2.

30. Use of an agonist of the cell surface receptor CD36 as expressed on mammalian dendritic cells in the manufacture of a medicament to induce a state of immune tolerance in a human.

31. The use as claimed in claim 30 wherein said CD36 agonist is selected from: an antibody with an affinity for an epitope of CD36, the Plasmodium falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1 for CD36 or thrombospondin, thrombospondin, apoptotic cells or a negatively charged phospholipid.

32. The use as claimed in claim 31 wherein the pf-EMP-1 active binding domain comprises the amino acid sequence as shown in Figure 2.

33. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD51 as expressed by mammalian dendritic cells and a pharmacologically acceptable carrier or diluent.

34. A composition as claimed in claim 33 suitable for inducing immune tolerance in a human wherein said CD51 agonist is selected from: an antibody with an affinity for an epitope of CD51, thrombospondin, apoptotic cells or a negatively charged phospholipid.

35. A composition as claimed in claim 33 which comprises the Plasmodium falciparum protein pf-EMP-1 or a protein comprising an active binding domain thereof and thrombospondin.

36. A composition as claimed in claim 35 wherein said active binding domain of pf-EMP-1 comprises the amino acid sequence shown in Figure 2.

37. Use of an agonist of the cell surface receptor CD51 as expressed on mammalian dendritic cells in the manufacture of a medicament for inducing a state of immune tolerance in a human.

38. Use of an agonist as claimed in claim 37 wherein said CD 51 agonist is selected from: an antibody with an affinity for an epitope of CD51, thrombospondin, apoptotic cells or a negatively charged phospholipid.

39. The use of an agonist as claimed in claim 37 wherein said medicament comprises, in combination, the Plasmodium falciparum protein pf-EMP-1 or a protein comprising an active binding domain thereof and thrombospondin.

40. A method of identifying a molecule which is an agonist of cell surface receptors CD36 and/or CD51 and/or a receptor for thrombospondin as expressed on antigen-presenting cells of the mammalian immune system which method comprises:
a) exposing mammalian antigen-presenting cells to the molecule to be tested, b) exposing said cells to an immune stimulus and c) determining the response to said immune stimulus by said cells, wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule under test is a potential agonist of CD36 and/or CD51 and/or a thrombospondin receptor and wherein if said test molecule is found to be a potential agonist of CD36 and/or CD51 and/or a thrombospondin receptor, the method further comprises the step of exposing said molecule to a purified sample of CD36 and/or CD51 and/or thrombospondin and detecting any direct binding between said molecule and CD36 and/or CD51 and/or thrombospondin.

41. A method as claimed in claim 40 wherein said thrombospondin receptor is not CD47.

42. A method as claimed in claim 40 or claim 41 wherein said response is maturation of said antigen-presenting cell.

43. A method as claimed in any one of claims 40 to 42 wherein said antigen-presenting cell of the immune system is selected from a dendritic cell, a macrophage, a B-lymphocyte or a monocyte.

44. A method of treating mammalian antigen-presenting cells in vitro to induce immune tolerance therein which comprises exposing said cells to an agonist of the cell surface receptor for thrombospondin and/or an agonist for the cell surface receptors CD36 and/or CD51.

45. A method as claimed in claim 44 wherein said mammalian antigen presenting cell is a dendritic cell.

46. A method as claimed in claim 44 or claim 45 wherein said antigen-presenting cell is human or mouse.

47. A method as claimed in claim 44 or 45 wherein said antigen presenting cell is selected from a macrophage, a H-lymphocyte or a monocyte.

48. A method as claimed in any one of claims 44 to 47 wherein said antigen-presenting cells are exposed to an antibody to an epitope of a thrombospondin receptor.

49. An antigen-presenting cell preparation obtainable by the method of any one of claims 44 to 48 for use as a medicament.

50. As antigen-presenting cell preparation obtainable by the method of any one of claims 44 to 48 for use in inducing peripheral immune tolerance in a human.

51. A method of identifying a molecule which is an agonist of a .beta.-integrin associated with the cell surface receptor CD51 as expressed on antigen-presenting cells of the mammalian immune system which method comprises:
a) exposing mammalian antigen-presenting cells to the molecule to be tested, b) exposing said cells to an immune stimulus and c) determining the response to said immune stimulus by said cells, wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule is an agonist of a .beta.-integrin associated with the cell surface receptor CD51.

52. A method as claimed in 51 wherein said .beta.-integrin is .beta.3 or .beta.5.

53. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of a .beta.-integrin associated with the cell surface receptor CD51 as expressed on mammalian antigen-presenting cells and a pharmacologically acceptable carrier or diluent.

54. A pharmaceutical composition as claimed in claim 53 wherein the .beta.-integrin is .beta.3 or .beta.5.

55. A method of inducing a state of immune tolerance in antigen-presenting cells of the mammalian immune system which comprises exposing said cells ex-vivo to an agonist of a .beta.-integrin.

56. A method as claimed in claim 55 wherein said .beta.-integrin is .beta.3 or .beta.5.

57. A method as claimed in claim 55 or 56 wherein said antigen presenting cells are human.

58. A method as claimed in any one of claims 55 to 57 wherein said cells are exposed to an antigenic material selected from an auto-antigen associated with a particular auto-immune disease, an allo-antigen, a xeno-antigen or a therapeutic substance which is likely to induce an unwanted immune response.

59. A preparation of cells obtainable by the method as claimed in any one of claims 55 to 58 for use as a medicament.

60. A preparation of cells obtainable by the method as claimed in any one of claims 54 to 57 for use in inducing immune-tolerance in a human.

61. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of a thrombospondin receptor and a pharmacologically acceptable carrier or diluent.

62. A composition as claimed in claim 61 wherein said thrombospondin receptor is not CD47.

63. A preparation of apoptotic cells for use in inducing peripheral immune tolerance in a mammal.

64. A preparation as claimed in claim 63 wherein said mammal is a human.

65. Use of a preparation comprising a negatively charged phospholipid in the manufacture of a medicament for use in inducing peripheral immune tolerance in a mammal.

66. The use as claimed in claim 65 wherein said mammal is a human.

67. The use as claimed in claim 65 or 66 wherein said medicament comprises liposomes including a negatively charged phospholipid.

68. The use as claimed any one of claims 65 to 67 wherein said negatively charged phospholipid is phosphatidylserine.

69. A method of inducing a state of immune tolerance in antigen-presenting cells of the mammalian immune system which comprises exposing said cells ex-vivo to a composition or preparation as defined in any one of claims 61 to 68.

70. A method as claimed in claim 69 wherein said cells are exposed to an antigenic material selected from: an auto-antigen associated with a particular auto-immune disease, an allo-antigen, a xeno-antigen or a therapeutic substance which is likely to induce an unwanted immune response.

71. A preparation of antigen-presenting cells obtainable by the method of claims 69 or 70.

72. A method of treating a human to induce peripheral immune tolerance therein comprising administering to said human a substance selected from the group consisting of: an agonist of CD36, an agonist of CD51, antagonist of a thrombospondin receptor, an agonist of a .beta.-integrin and a preparation of cells of any of claims 8, 9, 49, 50, 59, 60 or 71.

73. A method of identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells which comprises:
a) exposing immature human dendritic cells to the Plasmodium falciparum protein pf-EMP-1 or an active binding domain thereof in the presence or absence of the molecule to be tested, b) exposing said immature dendritic cells to an immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, wherein any maturation of said dendritic cells in the presence of the test molecule over and above that manifested in the absence of said molecule is an indication that said molecule is capable of preventing adherence of red blood cells infected with a malarial parasite to human dendritic cells.

74. A method as claimed in claim 73 wherein maturation of said dendritic cells is determined by examining the antigen-presenting ability of said cells.

75. A method as claimed in claim 73 or claim 74 wherein maturation of said dendritic cells is determined by examining said cells for expression of at least one cell surface antigen whose expression level is enhanced in response to an immune stimulus.

76. A method as claimed in claim 75 wherein maturation of said dendritic cells is determined by measuring the level of expression of two or more of the following panel of antigens:
HLA DR, CD54, CD40, CD83 and CD86.

77. A method as claimed in claim 76 wherein said cells are also examined for expression of CD80.

78. A method as claimed in any one of claims 75 to 77 wherein the level of expression of said antigen is detected using a labelled antibody.

79. A method as claimed in claim 73 wherein maturation is determined by measuring said cells' ability to induce T-cell proliferation.

80. A method as claimed in claim 73 wherein maturation of said dendritic cells is determined by quantifying the level of cytokines secreted from said cells.

81. A method as claimed in claim 80 wherein the level of secretion of TNF.alpha., IL12P70 and IL10 is measured.

82. A method as claimed in any one of claims 73 to 81 wherein said immune stimulus is lipopolysaccharide, TNF.alpha., CD40L or monocyte conditioned medium (MCM).